High-activity mutants of butyrylcholinesterase for cocaine hydrolysis and method of generating the same

ABSTRACT

A novel computational method and generation of mutant butyrylcholinesterase for cocaine hydrolysis is provided. The method includes molecular modeling a possible BChE mutant and conducting molecular dynamics simulations and hybrid quantum mechanical/molecular mechanical calculations thereby providing a screening method of possible BChE mutants by predicting which mutant will lead to a more stable transition state for a rate determining step. Site-directed mutagenesis, protein expression, and protein activity is conducted for mutants determined computationally as being good candidates for possible BChE mutants, i.e., ones predicted to have higher catalytic efficiency as compared with wild-type BChE. In addition, mutants A199S/A328W/Y332G, A199S/F227A/A328W/Y332G, A199S/S287G/A328W/Y332G, A199S/F227A/S287G/A328W/Y332G, and A199S/F227A/S287G/A328W/E441D all have enhanced catalytic efficiency for (−)-cocaine compared with wild-type BChE.

FIELD OF THE INVENTION

The present invention relates to butyrylcholinesterase variantpolypeptides, and in particular, butyrylcholinesterase mutants havingamino acid substitutions.

BACKGROUND OF THE INVENTION

Cocaine abuse is a major medical and public health problem thatcontinues to defy treatment. The disastrous medical and socialconsequences of cocaine addiction, such as violent crime, loss inindividual productivity, illness and death, have made the development ofan effective pharmacological treatment a high priority. However, cocainemediates its reinforcing and toxic effects by blocking neurotransmitterreuptake and the classical pharmacodynamic approach has failed to yieldsmall-molecule receptor antagonists due to the difficulties inherent inblocking a blocker. An alternative to receptor-based approaches is tointerfere with the delivery of cocaine to its receptors and accelerateits metabolism in the body.

The dominant pathway for cocaine metabolism in primates isbutyrylcholinesterase (BChE)-catalyzed hydrolysis at the benzoyl estergroup (Scheme 1).

Scheme 1. Schematic representation of BChE-catalyzed hydrolysis at thebenzoyl ester group.

Only 5% of the cocaine is deactivated through oxidation by the livermicrosomal cytochrome P450 system. Cocaine hydrolysis at benzoyl estergroup yields ecgonine methyl ester, whereas the oxidation producesnorcocaine. The metabolite ecgonine methyl ester is a biologicallyinactive metabolite, whereas the metabolite norcocaine is hepatotoxicand a local anesthetic. BChE is synthesized in the liver and widelydistributed in the body, including plasma, brain, and lung. Extensiveexperimental studies in animals and humans demonstrate that enhancementof BChE activity by administration of exogenous enzyme substantiallydecreases cocaine half-life.

Enhancement of cocaine metabolism by administration of BChE has beenrecognized to be a promising pharmacokinetic approach for treatment ofcocaine abuse and dependence. However, the catalytic activity of thisplasma enzyme is three orders-of-magnitude lower against the naturallyoccurring (−)-cocaine than that against the biologically inactive(+)-cocaine enantiomer. (+)-cocaine can be cleared from plasma inseconds and prior to partitioning into the central nervous system (CNS),whereas (−)-cocaine has a plasma half-life of approximately 45-90minutes, long enough for manifestation of the CNS effects which peak inminutes. Hence, BChE mutants with high activity against (−)-cocaine arehighly desired for use in humans. Although some BChE mutants withincreased catalytic activity over wild-type BChE have previously beengenerated, there exists a need for mutant BChE with even highercatalytic activity. Thus, prior mutants provide limited enhancement incatalytic activity over wild-type BChE.

Previous studies such as (a) Masson, P.; Legrand, P.; Bartels, C. F.;Froment, M-T.; Schopfer, L. M.; Lockridge, O. Biochemistry 1997, 36,2266 (b) Masson, P.; Xie, W., Froment, M-T.; Levitsky, V.; Fortier,P.-L.; Albaret, C.; Lockridge, O. Biochim. Biophys. Acta 1999, 1433,281, (c) Xie, W.; Altamirano, C. V.; Bartels, C. F.; Speirs, R. J.;Cashman, J. R.; Lockridge, O. Mol. Pharmacol. 1999, 55, 83, (d) Duysen,E. G.; Bartels, C. F.; Lockridge, O. J. Pharmacol. Exp. Ther. 2002, 302,751, (e) Nachon, F.; Nicolet, Y.; Viguie, N.; Masson, P.;Fontecilla-Camps, J. C.; Lockridge, O. Eur. J. Biochem. 2002, 269, 630,(f) Zhan, C.-G.; Landry, D. W. J. Phys. Chem. A 2001, 105, 1296;Berkman, C. E.; Underiner, G. E.; Cashman, J. R. Biochem. Pharmcol.1997, 54, 1261; (g) Sun, H.; Yazal, J. E.; Lockridge, O.; Schopfer, L.M.; Brimijoin, S.; Pang, Y.-P. J. Biol. Chem. 2001, 276, 9330, (h) Sun,H.; Shen, M. L.; Pang, Y. P.; Lockridge, O.; Brimijoin, S. J. Pharmacol.Exp. Ther. 2002, 302, 710, (i) Sun, H.; Pang, Y. P.; Lockridge, O.;Brimijoin, S. Mol. Pharmacol. 2002, 62, 220 (hereinafter “Sun et al”);and (j) Zhan, C.-G.; Zheng, F.; Landry, D. W. J. Am. Chem. Soc. 2003,125, 2462 (hereinafter “Zhan et al”), herein all incorporated byreference, suggested that, for both (−)-cocaine and (+)-cocaine, theBChE-substrate binding involves two different types of complexes:non-prereactive and prereactive BChE-substrate complexes. Whereas thenon-prereactive BChE-cocaine complexes were first reported by Sun et al,Zhan et al were the first reporting the prereactive BChE-cocainecomplexes and reaction coordinate calculations, disclosed in Zhan et al.It was demonstrated that (−)/(+)-cocaine first slides down thesubstrate-binding gorge to bind to W82 and stands vertically in thegorge between D70 and W82 (non-prereactive complex) and then rotates toa position in the catalytic site within a favorable distance fornucleophilic attack and hydrolysis by S198 (prereactive complex). In theprereactive complex, cocaine lies horizontally at the bottom of thegorge. The main structural difference between the BChE-(−)-cocainecomplexes and the corresponding BChE-(+)-cocaine complexes exists in therelative position of the cocaine methyl ester group. Reaction coordinatecalculations revealed that the rate-determining step of BChE-catalyzedhydrolysis of (+)-cocaine is the chemical reaction process, whereas for(−)-cocaine the change from the non-prereactive complex to theprereactive complex is rate determining. A further analysis of thestructural changes from the non-prereactive complex to the prereactivecomplex reveals specific amino acid residues hindering the structuralchanges, providing initial clues for the rational design of BChE mutantswith improved catalytic activity for (−)-cocaine.

Previous molecular dynamics (MD) simulations of prereactive BChE-cocainebinding were limited to wild-type BChE. Even for the non-prereactiveBChE-cocaine complex, only one mutant (A328W/Y332A) BChE binding with(−)-cocaine was simulated and its catalytic activity for (−)-cocaine wasreported by Sun et al. No MD simulation was performed on any prereactiveenzyme-substrate complex for (−)- or (+)-cocaine binding with a mutantBChE. In addition, all previous computational studies of Sun et al andZhan et al of BChE interacting with cocaine were performed based on ahomology model of BChE when three-dimensional (3D) X-ray crystalstructure was not available for BChE, as taught by Nicolet, Y.;Lockridge, O.; Masson, P.; Fontecilla-Camps, J. C.; Nachon, F. J. Biol.Chem. 2003, 278, 41141 (hereinafter “Nicolet et al”), recently reported3D X-ray crystal structures of BChE. As expected, the structure of BChEis similar to a previously published theoretical model of this enzymeand to the structure of acetylcholinesterase.

The main difference between the experimentally determined BChE structureand its model was found at the acyl binding pocket (acyl loop) that issignificantly bigger than expected. It is unclear whether the structuraldifference at the acyl binding pocket significantly affect BChE bindingwith (−)-cocaine and (+)-cocaine. Although previous MD simulations ofcocaine binding with wild-type BChE and the reaction coordinatecalculations point to some amino acid residues that might need to bemutated for the purpose of improving the catalytic activity for(−)-cocaine hydrolysis, it remained unknown which exact amino acidmutations will result in a BChE with a higher catalytic activity for(−)-cocaine.

Computational studies of wild-type BChE and cocaine from Sun, et al,based on a “homology model,” suggest that the rate-determining step forBChE-catalyzed hydrolysis of cocaine is the rotation of the cocaine inthe active site of BChE. By decreasing the hindrance of the rotation,the rate of the hydrolysis may be enhanced. Sun, et al describescreating an A328W/Y332A BChE mutant by: (1) replacing Tyr332 with Ala,“to reduce the steric hindrance and the p-p interaction that impederotation,” and (2) replacing Ala328 with Trp “to provide a cation-pinteraction to restore substrate affinity lost in disabling the p-pinteraction.”

Sun et al studied the A328W/Y332A BChE mutant using enzyme assays andkinetics. In vitro studies were conducted using human plasma and in vivostudies were conducted using male Sprague-Dawley rats. The mutant wasfound to have enhanced catalytic properties. The mutant was furtherstudied using molecular modeling. The three dimensional (3D) structureof A328W/Y332A was generated from the computationally generated 3D modelof wild-type BChE and changing the relevant residues using commerciallyavailable software. Cocaine was docked to the catalytic gorge of themutant BChE using other commercially available software. Thecocaine-enzyme complex was refined by molecular dynamic simulation. Thedata generated by the molecular modeling studies were consistent withenzyme assays and kinetic data.

It should be noted that all prior computational techniques (moleculardocking and molecular dynamics simulation) used by other researchers arebased on an empirical force field which cannot be used to perform anynecessary reaction coordinate calculation for the detailed understandingof the complicated catalytic reaction process. As it is well-known, itis particularly challenging to model and simulate the detailed reactionpathway and predict the kinetics of such an enzymatic reaction.

U.S. Patent Application Publication Nos. 2004/0121970; 2004/0120939; and2003/0153062, describe 20+BChE mutants, or “variants,” from human andother animals, each having from one to six amino acid alterations andincreased cocaine hydrolysis activity. For example, mutants includeF227A/A328W; F227A/S287G/A328W; A119S/S287G/A328W;A328W/Y332M/S287G/F227A, A199S/F227A/S/287G/A328W andA119S/F227A/S287G/A328W/Y332M. The mutants have varying increases incatalytic activity, up to 100-fold increase relative to wild-type BChE.

There exists a need in the art for determining which proposed mutantBChEs should have ever increasing catalytic activity and for generatingthose mutants which should have enhanced catalytic activity.

SUMMARY OF THE INVENTION

The present invention includes five novel human BChE mutants that haveunexpected increased catalytic efficiency for cocaine hydrolysis. Themutants have various unique amino acid residue substitutions whichprovide the surprising enhanced catalytic activity. These mutants are(1) A199S/A328W/Y332G mutant (SEQ ID NO: 2), which has a approximately65-fold improved catalytic efficiency against (−)-cocaine; (2)A199S/F227A/A328W/Y332G mutant (SEQ ID NO: 8), which has anapproximately 148-fold improved catalytic efficiency against(−)-cocaine; (3) A199S/S287G/A328W/Y332G mutant (SEQ ID NO: 14), whichhas an approximately 456-fold improved catalytic efficiency against(−)-cocaine; (4) A199S/F227A/S287G/A328W/Y332G mutant (SEQ ID NO: 20),which has an approximately 1,003-fold improved catalytic efficiencyagainst (−)-cocaine; and (5) A199S/F227A/S287G/A328W/E441D mutant (SEQID NO: 26), which has an approximately 445-fold improved catalyticefficiency against (−)-cocaine.

In addition, the aforementioned mutant amino acid sequences can betruncated without substantially affecting the catalytic activity so thatamino acid residues 1-67 and 443-574 can be removed withoutsubstantially affecting the catalytic activity of the enzyme. SEQ IDNOS: 4, 10, 16, 22 and 28 are the amino acid sequences for residue68-142 corresponding to mutants 1-5, respectively. In addition, withregard to mutants 1-4, it was found that amino acid residues before 117and after 438 could be removed without substantially changing theactivity of the mutant enzymes, resulting in truncated amino acidsequences having SEQ ID NOS: 6, 12, 18 and 24, respectively. Finallywith regard to mutant 5, amino acid residues before 117 and after 441could be removed without substantially changing its activity resultingin SEQ ID NO: 30.

These aforementioned truncated sequences all of with similar catalyticactivity is based on protein structures.

Further, the present invention is directed to a novel and uniquepharmaceutical composition which comprises a butyrylcholinesterasevariant, namely mutants 1-5, along with a suitable pharmaceuticalcarrier. The pharmaceutical composition can be administered to anindividual in an effective amount to lower the patient's cocaine bloodconcentration and in particular (−)-cocaine blood concentration.

In addition, the present invention is directed to a novel and uniquemethod for developing mutants which have enhanced catalytic efficiency.The generation method includes both a computational portion and anexperimental portion. With regard to the computational portion, avariety of state of the art computational techniques including molecularmodeling, molecular dynamics (MD) simulations and hybrid quantummechanical/molecular mechanical (QM/MM) calculations, provide a virtualscreening of possible BChE mutants. This virtual screening predictswhich mutation will lead to a more stable transition state for arate-determining step compared to the corresponding separated reactants,i.e., free cocaine and free enzyme. The more stable the transitionstate, the lower the energy barrier, and the higher the catalyticefficiency. Following the computational portion, an experimental test isthen conducted on the possible mutants of the computation portion. Theexperimental test includes site-directed mutagenesis, proteinexpression, and enzyme activity assay. The experimental tests areconducted on mutants which are predicted to have a high catalyticefficiency against (−)-cocaine than the wild-type BChE and/or otherknown BChE mutants against (−)-cocaine. Thus, the present methodidentifies or predicts mutants having high catalytic activity forcocaine hydrolysis by performing molecular modeling and MD simulationson the transition state structures of possible mutants of BChE. Thismethod is an improvement over traditional random-search approaches,which, given the complex catalytic mechanism of cocaine hydrolysis,makes it difficult to improve the catalytic activity of BChE for cocainehydrolysis.

The present invention in one form, concerns a butyrylcholinesterasevariant peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, and 30.

The present invention in another form thereof concerns a nucleic acidmolecule comprising a nucleic acid sequence which encodes abutyrylcholinesterase variant peptide, the nucleic acid sequenceselected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, and 29.

The present invention in another form thereof concerns a pharmaceuticalcomposition comprising a butyrylcholinesterase variant polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOS. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30;and a suitable pharmaceutical carrier.

The present invention in another form thereof concerns a method fortreating a cocaine-induced condition comprising administering to anindividual an effective amount of butyrylcholinesterase variant peptidehaving an amino acid sequence selected from the group consisting of SEQID NOS. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, tolower blood cocaine concentration.

The present invention is another form thereof concerns a method fortreating a cocaine induced condition comprising administering to anindividual an effective amount of a pharmaceutical compositioncomprising a butyrylcholinesterase variant having an amino acid sequenceselected from the group consisting of SEQ ID NOS. 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, and 30, and suitable pharmaceuticalcarrier of claim 3 to an individual in an effective amount to lowerblood cocaine concentration.

The present invention in yet another form thereof concerns a method forgenerating butyrylcholinesterase mutants. The method includes generatingan initial structure of the transition state structure for therate-determining step of the cocaine hydrolysis catalyzed by a possiblebutyrylcholinesterase mutant. A sufficiently long time moleculardynamics simulation is performed on the transition state structure inwater to have a stable molecular dynamics trajectory. The moleculardynamics trajectory is analyzed and the hydrogen bonding energies areestimated between the carboxyl oxygen of the (−)-cocaine benzyl esterand the oxyanion hole of the possible butyrylcholinesterase mutant. Ifthe overall hydrogen binding energy between the carboxyl oxygen of the(−)-cocaine benzyl ester and the possible butyrylcholinesterase mutant,in the transition state, is stronger than the overall hydrogen bindingenergy between the carboxyl oxygen of the (−)-cocaine benzyl ester andthe wild-type butyrylcholinesterase, optionally, hybrid quantummechanical/molecular mechanical (QM/MM) geometry optimization isperformed to refine the molecular dynamics-simulated structure, thehydrogen binding energies are calculated and the energy barrier isevaluated. Finally, the butyrylcholinesterase mutant is generated.

In various alternative embodiments, the generating an initial structureof the transition state structure is based on reaction coordinatecalculations for the wild-type butyrylcholinesterase. The generatingbutyrylcholinesterase mutant includes performing site-directedmutagenesis on a nucleic acid sequence which includes wild-typebutyrylcholinesterase to generate the mutant butyrylcholinesterasenucleic acid sequence. Using the mutant butyrylcholinesterase nucleicacid sequence, the protein encoded by the mutant nucleic acid sequencesis expressed to produce mutant butyrylcholinesterase and catalyticactivity assay is performed on the mutant butyrylcholinesterase.

The hybrid quantum mechanical/molecular mechanical geometry optimizationmay include calculating the hydrogen binding energies and evaluating theenergy barriers only if the overall hydrogen binding energy between thecarboxyl oxygen of the (−)-cocaine benzyl ester and the possiblebutyrylcholinesterase mutant, in the transition state, is stronger thanknown butyrylcholinesterase mutants against (−)-cocaine.

In yet another alternative further embodiment, the method for generatingbutyrylcholinesterase mutants further includes determining therate-limiting step in the hydrolysis of (−)-cocaine by the possiblebutyrylcholinesterase mutant by conducting molecular dynamicssimulations and quantum mechanical/molecular mechanical calculationsrelating to the transition states for other reaction steps between(−)-cocaine by the possible butyrylcholinesterase mutant and calculatingrespective energy barriers, thereby establishing which of the reactionsteps is the rate-determining one.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 are plots of distances in the MD simulation (−)-cocaine bindingwith A328W/Y332G BChE versus the simulation time, along withroot-mean-square deviation (RMSD) in the enzyme-substrate complexeswhere FIG. 1A represents the non-prereactive enzyme-substrate complexes;and FIG. 1B represents the prereactive enzyme-substrate complexes inaccordance with the present invention.

FIG. 2 shows the binding of structures of the simulated non-prereactiveand prereactive complexes of wild-type BChE binding with two entomers ofcocaine in which FIG. 2A depicts BChE (−)-cocaine non-prereactivecomplex; FIG. 2B depicts BChE (−)-cocaine prereactive complex, FIG. 2Cdepictes BChE (+)-cocaine non-prereactive complex; and FIG. 2D depictsBChE (+)-cocaine prereactive complex.

FIG. 3A shows the (−)-cocaine rotation in the BChE active site for thenon-prereactive complex to the prereactive complex hindered by someresidues at positions Y332, A328, and F329 residues in thenon-prereactive complexes which are significantly different from thecorresponding positions in the prereactive complex; and FIG. 3B showsthe (+)-cocaine rotation in the BChE active site where none of theaforementioned residues hinders the (+)-cocaine rotation in the BChEactive site from the non-prereactive complex to the prereactive complexin accordance with the present invention.

FIG. 4A depicts the (−)-cocaine rotation in the active site ofA328W/Y332A; FIG. 4B depicts the (−)-cocaine rotation in the active siteof A328W/Y332G BChE from the non-preactive complex to the prereactivecomplex; and FIG. 4C depicts the (−)-cocaine rotation in the active siteof wild-type BChE.

FIG. 5A is a plot of the key internuclear distances (in Å) versus thetime in the simulated TS1 structure for (−)-cocaine hydrolysis catalyzedby A328W/Y332A; and FIG. 5B for A199S/A328W/Y332G BChE.

FIG. 6 is a plot of key internuclear distances (in A) versus the time inthe simulated TS1 structure for (−)-cocaine hydrolysis catalyzed byA199S/S287G/A328W/Y332G BChE.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention has two major improvements over the prior art. Thefirst is the presently discovered BChE mutants, mutant 1,A199S/A328W/Y332G; mutant 2, A199S/F227A/A328W/Y332G; mutant 3,A199S/S287G/A328W/Y332G; mutant 4, A199S/F227A/S287G/A328W/Y332G; andmutant 5, A199S/F227A/S287G/A328W/E441 D each have a significantlyhigher catalytic efficiency. The second improvement is concerning themutant designing or discovering process.

The BChE mutants 1-5 have full length amino acid sequences, SEQ ID NOS:2, 8, 14, 20, and 26, respectively, which are encoded by nucleic acidsequences having SEQ ID NOS: 1, 7, 13, 19, and 25, respectively. Table 1summarizes the catalytic efficiency against (−)-cocaine for the fivemutants.

In addition to the full length BChE mutants, the respective amino acidsequence can be truncated without substantially affecting the respectivecatalytic activity. With all mutants, residues 1-67 and 443-574 can beremoved without substantially affecting the catalytic activity of therespective mutant BChE. Further, with regard to mutant 1-4, amino acids1-116 and 439-574 can be omitted without substantially affecting itsrespective catalytic activity. With regard to mutant 5, amino acidresidues 1-116 and 442-574 can be omitted without substantiallyaffecting its catalytic activity. Table 1 also provides a summary ofamino acid SEQ ID NOS and corresponding nucleic acid SEQ ID NOS for theaforementioned truncated mutant BChE sequences.

TABLE 1 Partially Truncated Nucleic Acid SEQ ID NO SequenceCorresponding SEQ ID NO. Catalytic Corresponding SEQ ID NO. To AminoAcid for Amino Efficiency Mutant Amino Acid Nucleic Acid Amino Acid ToAmino Acid for Amino Acid Residues Acid Residues Against NumberSubstituting SEQ ID NO. SEQ ID NO. Residues 68-442 Residues 68-442117-438/441* 117-438/441* (−)-cocaine 1 A199S/A328W/ 1 2 3 4 5 6 65-fold Y332G 2 A199S/F227A/ 7 8 9 10 11 12 148-fold A328W/Y332G 3A199S/S287G/ 13 14 15 16 17 18 456-fold A328W/Y332G 4 A199S/F227A/ 19 2021 22 22 24 1,003-fold  S287G/A328W/ Y332G 5 A199S/F227A/ 25 26 27 28 2930 445-fold S287G/A328W/ E441D (*Amino acid residues 117-438 for mutants1-4 and residues 117-441 for mutant 5.)

The BChE variant polypeptide, e.g., SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, and 30 can be formulated in a pharmaceuticalcomposition along with a suitable pharmaceutical carrier known to oneskilled in the art.

The present BChE variant polypeptides can be used in treating acocaine-induced condition by administering to an individual, aneffective amount of one of the BChE variant polypeptides, i.e., SEQ IDNOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, tolower blood cocaine concentration. The BChE variant polypeptide may beadministered in the form of a pharmaceutical composition in which theBChE variant is included with a suitable pharmaceutical carrier.Treatment of a cocaine induced condition using one of the aforementionedBChE variant polypeptides can be done in accordance with Zhan et al.,page 2463.

The preferred dose for administration of a butyrylcholinesterase orpeptide composition in accordance with the present invention is thatamount which will be effective in lowering (−)-cocaine concentration ina patient's bloodstream, and one would readily recognize that thisamount will vary greatly depending on the nature of cocaine consumed,e.g., injected or inhaled, and the condition of a patient. An “effectiveamount” of butyrylcholinesterase mutant or pharmaceutical agent to beused in accordance with the invention is intended to mean a nontoxic butsufficient amount of the agent, such that the desired prophylactic ortherapeutic effect is produced. Thus, the exact amount of the enzyme ora particular agent that is required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the condition being treated, the particular carrier oradjuvant being used and its mode of administration, and the like.Similarly, the dosing regimen should also be adjusted to suit theindividual to whom the composition is administered and will once againvary with age, weight, metabolism, etc. of the individual. Accordingly,the “effective amount” of any particular butyrylcholinesterasecomposition will vary based on the particular circumstances, and anappropriate effective amount may be determined in each case ofapplication by one of ordinary skill in the art using only routineexperimentation.

A unique method was used to determine potential BChE mutants withprojected increased catalytic activity for the hydrolysis of cocaine.The method provides a unique approach which first models the potentialBChE mutant interaction with cocaine followed by generating the BChEmutant if the predicted model indicates that the BChE variant shouldhave enhanced catalytic activity. The method includes generating aninitial structure of the transition state structure for therate-determining step for the cocaine hydrolysis catalyzed by a possibleBChE mutant. A sufficiently long time molecular dynamics simulation isperformed on the transition state structure in water to have a stablemolecular dynamics trajectory. The molecular dynamics trajectory isanalyzed and the hydrogen binding energies are estimated between thecarboxyl oxygen of the (−)-cocaine benzoyl ester and the oxyanion holeof the possible BChE mutant. If the overall hydrogen binding energybetween the carboxyl oxygen of the (−)-cocaine benzoyl ester and thepossible BChE mutant, in the transition state, is stronger than theoverall hydrogen binding energy between the carboxyl oxygen of the(−)-cocaine benzoyl ester and the wild-type BChE, hybrid quantummechanical/molecular mechanical (QM/MM) geometry optimization isperformed to refine the molecular dynamics-simulated structure, thehydrogen binding energies are calculated and the energy barrier isevaluated. The QM/MM calculations make the computational predictionsmore reliable. Finally, the BChE mutant is generated.

With regard to the molecular dynamics (MD) simulations and quantummechanical/molecular mechanical (QM/MM) calculations, the first chemicalreaction step of (−)-cocaine hydrolysis catalyzed bybutyrylcholinesterase (BChE) mutants and, when needed, other reactionsteps are modeled and calculated using molecular dynamics simulationsand QM/MM calculations. Following this modeling, mutant BChE's arecreated using site-directed mutagenesis followed by protein expression.The aforementioned five mutants were identified by computationalanalysis and generated by site-directed mutagenesis which havesignificantly enhanced (−)-cocaine hydrolysis catalytic efficiencycompared with wild-type BChE.

In the present method computational analysis in the form of molecularmodeling of a potential BChE mutant and MD simulations and QM/MMcalculations provide virtual screening of possible BChE mutants whichhave predicted enhanced catalytic activity for (−)-cocaine. For example,the MD simulations and QM/MM calculations predict which mutation willlead to a more stable transition state for the rate determining stepcompared to the corresponding separated reagents, i.e., free cocaine andfree possible mutant BChE, where the more stable transition state leadsto a lower energy barrier and higher predicted catalytic efficiency.Only after the computational analysis predicts enhanced catalyticefficiency, is site-directed mutagenesis conducted on wild-type BChEnucleic acid sequence to generate a mutant nucleic acid sequence whichis then used to express a mutant BChE protein. The mutant BChE proteinis then used in catalytic assays to determine the catalytic efficiencyagainst (−)-cocaine.

The use of predictive, computational modeling of the present method foridentifying mutant BChE candidates and the resulting mutant BChE arenovel and unexpected over prior conventional methods which will now bereadily apparent to one of ordinary skill in the art.

Using the present method, most discovered new mutants include a specificmutation (Y332G) on residue #332. No prior BChE mutant having the Y332Gmutation had ever been reported previously; only mutations Y332A andY332M on residue #332 had been tested previously by other researchers.Prior to the present invention, there was no reason to expect that amutant including Y332G mutation should be better than the correspondingmutant including Y332A mutation or Y332M. Thus, the present mutants witha Y332G mutation which have enhanced catalytic activity represent asurprising and unexpected result over prior BChE mutants.

A Y332G mutant (single mutation) was first tested and found that theY332G mutant had a slightly low (or approximately equal) catalyticefficiency than the wild-type. So, only an appropriate combination ofdifferent mutations on different residues could make the enzyme moreactive. As seen below, the prior art did not reveal that any of theparticular combinations tested was expected to have an improvedcatalytic efficiency. The present method is based on the present unique,extensive computational modeling and simulations of the detailedcatalytic mechanism for both the wild-type BChE and the mutants.

The primary improvement of the present method over the prior art is thathigh-performance computational modeling and simulations of the detailedcatalytic mechanism are performed, which includes modeling how cocainebinds with BChE and the subsequent structural transformation andchemical reaction process. The prior art only considered the cocainebinding with the enzyme (BChE) and was unable to examine the detailedcatalytic reaction process after the BChE-cocaine binding. Whenmolecular modeling was limited to studying the BChE-cocaine binding, onecould only design a mutation to improve the BChE-cocaine binding withoutknowing whether the mutation will also speedup the subsequent chemicalreaction process or not.

To overcome the obstacles of prior challenges to using computationaltechniques such as molecular docking and molecular dynamics simulationpreviously used by others which were based on an empirical force fieldwhich cannot be used to perform necessary reaction coordinatecalculations for the catalytic reaction process, a variety ofstate-of-the-art computational techniques of homology modeling,molecular docking, molecular dynamics, quantum mechanics (QM), andhybrid quantum mechanics/molecular mechanics (QM/MM) were used for therational design of the BChE mutants. The combined use of thesecomputational techniques, including QM and QM/MM, led to the study ofthe detailed reaction coordinate of the BChE-catalyzed hydrolysis ofcocaine which, for the first time, provided the detailed structures ofall transition states and intermediates existing in the reaction processand the corresponding energetics. These extensive computational modelingand simulation studies provided for the rational design of possible BChEmutants that not only can improve the BChE-cocaine binding, but also canspeedup the subsequent chemical reaction process. As a result, one cannow quickly discover the BChE mutants with the significantly improvedcatalytic efficiency.

In addition, to the differences mentioned above, the present molecularmodeling of the BChE-cocaine binding also differs from the priormodeling. The molecular modeling in the prior art considered only onebinding mode for each BChE-cocaine system, without modeling the possiblecocaine rotation in the BChE active site after the binding. The presentmethod considers two different binding modes for each BChE-cocainesystem and the structural transformation between them: non-prereactiveand prereactive BChE-cocaine complexes. The present modeling providesmore detailed information about the BChE-cocaine binding and thesubsequent structural transformation.

The present method includes molecular dynamics simulations performed onthe cocaine binding with both the wild-type BChE and the mutants whereasprior molecular dynamics simulations were only performed on the cocainebinding with the wild-type BChE. As is shown below in the followingexperiments, the computational prediction could be completely wrongwithout directly modeling and simulating cocaine binding with theproposed mutants.

The present invention will now be discussed with regard to the followingnon-limiting examples in the form of experiments which are provided toenhance understanding of the present invention but in no way limit itsscope or applicability.

Experiment 1 Computational Study of Cocaine Binding with Wild-Type andMutant BChE's for A328W/Y332G, A328W/Y332A, and A328W/Y332A/Y419S

A detailed computational study of cocaine binding with wild-type andmutant BChE's starting from the available X-ray crystal structure ofwild-type BChE was performed. The simulated mutants include A328W/Y332G,A328W/Y332A, and A328W/Y332A/Y419S, as simple geometric consideration ofthe binding site suggests that these mutations could be important forchanging the (−)-cocaine rotation from the non-prereactive complex tothe prereactive complex. Wet experimental tests were conducted on thecatalytic activity of these mutants for (−)-cocaine in order to verifythe computational predictions. All of the obtained results clearlydemonstrate that molecular modeling and MD simulations of cocainebinding with BChE mutants provide a reliable computational approach tothe rational design of high-activity mutants of BChE for the (−)-cocainehydrolysis.

3D model of BChE. The initial coordinates of human BChE used in thecomputational studies came from the X-ray crystal structure deposited inthe Protein Data Bank (pdb code: 1 POP). The missing residues (D2, D3,E255, D378, D379, N455, L530, E531, and M532) in the X-ray crystalstructure were built using the automated homology modeling tool Modelerdisclosed by Sali, A.; Blundell, T. L. J. Mol. Biol. 1990, 212 403, andSali, A.; Blundell, T. L. J. Mol. Biol. 1993, 234, 779, hereinincorporated by reference, and Insightll software (Accelrys, Inc.) withthe default parameters.

Molecular docking. Molecular docking was performed for eachnon-prereactive protein-ligand binding complex. The binding site wasdefined as a sphere with an approximately 15 Å radius around the activesite residue S198. The amino acid residues included in the binding sitemodel are not contiguous in the protein. Cocaine, considered as aligand, was initially positioned at 17 Å in front of S198 of the bindingsite. Each BChE-cocaine binding complex was energy-minimized by usingthe steepest descent algorithm first until the maximum energy derivativeis smaller than 4 kcal/mol/Å and then the conjugated gradient algorithmuntil the maximum energy derivative is smaller than 0.001 kcal/mol/Å.The energy minimization was followed by a 300 ps molecular dynamics (MD)simulation at T=298 K with a time step of 1 fs. During the energyminimization and MD simulation, only cocaine and the residues of BChEincluded in the binding site were allowed to move, while the remainingpart of the protein was fixed. The energy-minimization and MD simulationfor these processes were performed by using the Amber force fieldimplemented in the Discover_(—)3/InsightII calculation engine, disclosedby Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, Jr.,K. M.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.;Kollman, P. A. J. Am. Chem. Soc. 1995, 117, 5179. The non-bonded cut-offmethod and the dielectric constant were set up to group based (12 Åcut-off distance) and distance dependent, respectively (ε=4r) inaccordance with Harvey, S. C. Proteins 1989, 5, 78-92, hereinincorporated by reference.

Molecular dynamic simulation in water. The initial coordinates used inthe MD simulation of the non-prereactive complexes were determined byusing the molecular docking procedure described above, whereas theinitial coordinates used in the MD simulation of the prereactivecomplexes were obtained from superimposing backbone of the X-ray crystalstructure to that of the previously disclosed simulated prereactivecomplex of Zhan et al between cocaine and a homology model of wild-typeBChE. Each BChE-cocaine binding complex was neutralized by adding twochloride counterions and was solvated in a rectangular box of TIP3Pwater molecules with a minimum solute-wall distance of 10 Å. The generalprocedure for carrying out the MD simulations in water is similar tothat used in our previously reported other computational studies such asthose in Zhan et al and (a) Zhan, C.-G.; Norberto de Souza, O.;Riftenhouse, R.; Ornstein, R. L. J. Am. Chem. Soc. 1999, 121, 7279, (b)Koca, J.; Zhan, C.-G.; Rittenhouse, R.; Ornstein, R. L. J. Am. Chem.Soc. 2001, 123, 817, (c) Koca, J.; Zhan, C.-G.; Rittenhouse, R. C.;Ornstein, R. L. J. Comput. Chem. 2003, 24, 368, herein all incorporatedby reference. These simulations were performed by using the Sandermodule of Amber7 program as taught by Case, D. A.; Pearlman, D. A.;Caldwell, J. W.; Cheatham III, T. E.; Wang, J.; Ross, W. S.; Simmerling,C. L.; Darden, T. A.; Merz, K. M.; Stanton, R. V.; Cheng, A. L.;Vincent, J. J.; Crowley, M.; Tsui, V.; Gohlke, H.; Radmer, R. J.; Duan,Y.; Pitera, J.; Massova, I.; Seibel, G. L.; Singh, U. C.; Weiner, P. K.;Kollman, P. A. (2002), AMBER 7, University of California, San Francisco,herein incorporated by reference. The solvated system was optimizedprior to the MD simulation. First, the protein-ligand was frozen and thesolvent molecules with counterions were allowed to move during a5000-step minimization with the conjugate gradient algorithm and a 5 psMD run at T=300 K. After full relaxation and the entire solvated systemwas energy-minimized, the system was slowly heated from T=10 K to T=300K in 30 ps before the production MD simulation for 500 ps. The fullminimization and equilibration procedure was repeated for each mutant.The MD simulations were performed with a periodic boundary condition inthe NPT ensemble at T=300 K with Berendsen temperature coupling andconstant pressure (P=1 atm) with isotropic molecule-based scalingdisclosed in Berendsen, H. C.; Postma, J. P. M.; van Gunsteren, W. F.;DiNola, A.; Haak, J. R. J. Comp. Phys. 1984, 81, 3684, hereinincorporated by reference. The SHAKE algorithm of Ryckaert, J. P.;Ciccotti, G.; Berendsen, H. C. J. Comp. Phys. 1977, 23, 327 (hereinincorporated by reference) was applied to fix all covalent bondscontaining a hydrogen atom, a time step of 2 fs was used, and thenon-bond pair list was updated every 10 steps. The pressure was adjustedby isotropic position scaling. The particle mesh Ewald (PME) method ofEssmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T. A.; Lee, H.,Pedersen; L. G. J. Chem. Phys. 1995, 98, 10089, herein incorporated byreference, was used to treat long-range electrostatic interactions. Aresidue-based cutoff of 10 Å was applied to the noncovalentinteractions. During the 500 ps production MD simulation, thecoordinates of the simulated complex were saved every 1 ps.

Molecular docking and MD simulation procedures described above wereperformed to study cocaine binding with wild-type BChE and threemutants, i.e., A328W/Y332A, A328W/Y332A/Y419S, and A328W/Y332G. For eachprotein system (wild-type or mutant BChE), the protein binding withcocaine was considered in both the non-prereactive and prereactiveenzyme-substrate complexes.

Most of the MD simulations in water were performed on a supercomputer,Superdome (shared-memory, with 4 nodes and 256 processors), at theCenter for Computational Sciences, University of Kentucky. The othercomputations were carried out on SGI Fuel workstations and a34-processors IBM x335 Linux cluster.

Experimental procedure. Site-directed mutagenesis of human BChE cDNA wasperformed by the QuikChange method of Braman, J.; Papworth, C.; Greener,A. Methods Mol. Biol. 1996, 57, 5731, herein incorporated by reference.Mutations were generated from wild-type human BChE in a pRc/CMVexpression plasmid in accordance with Xie, W.; Altamirano, C. V.;Bartels, C. F.; Speirs, R. J.; Cashman, J. R.; Lockridge, O. Mol.Pharmacol. 1999, 55, 83, all herein incorporated by reference, kindlyprovided by Dr. Lockridge at University of Nebraska Medical Center.Using plasmid DNA as template and primers with specific base-pairalterations, mutations were made by polymerase chain reaction with PfuDNA polymerase, for replication fidelity. The PCR product was treatedwith Dpn I endonuclease to digest the parental DNA template. Modifiedplasmid DNA was transformed into Escherichia coli, amplified, andpurified. The DNA sequences of the mutants were confirmed by DNAsequencing. BChE mutants were expressed in human embryonic kidney cellline 293T/17. Cells were grown to 80-90% confluence in 6-well dishes andthen transfected by Lipofectamine 2000 complexes of 4 μg plasmid DNA pereach well. Cells were incubated at 37° C. in a CO₂ incubator for 24hours and cells were moved to 60-mm culture vessel and cultured for fourmore days. The culture medium [10% fetal bovine serum in Dulbecco'smodified Eagle's medium (DMEM)] was harvested for a BChE activity assay.To measure cocaine and benzoic acid, the product of cocaine hydrolysisby BChE, we used sensitive radiometric assays based on tolueneextraction of [³H]cocaine labeled on its benzene ring were used inaccordance with Masson, P.; Xie, W., Froment, M-T.; Levitsky, V.;Fortier, P.-L.; Albaret, C.; Lockridge, O. Biochim. Biophys. Acta 1999,1433, 281, herein incorporated by reference. In brief, to initiatereactions, 100 nCi of [³H]cocaine was mixed with 100 μl of culturemedium. Reactions proceeded at 37° C. for varying times. Reactions werestopped by adding 300 μl of 0.02 M HCl, which neutralized the liberatedbenzoic acid while ensuring a positive charge on the residual cocaine.[³H]benzoic acid was extracted by 1 ml of toluene and measured byscintillation counting. Finally, the measured time-dependent radiometricdata were fitted to the kinetic equation so that the catalyticefficiency (k_(cat)/K_(M)) was determined.

Depicted in FIG. 1 are plots of some important distances in theMD-simulated (−)-cocaine binding with A328W/Y332G BChE versus thesimulation time, along with root-mean-square deviation (RMSD) of thecoordinates of backbone atoms in the simulated structure from those inthe X-ray crystal structure. MD trajectories for other complexes weresimilar to these two in FIG. 1, although the simulated average distancesare different. Summarized in Table 2 are the average values of someimportant geometric parameters in the simulated complexes.

TABLE 2 Average values of the geometric parameters^(c) RMSD^(d)BChE-cocaine binding^(a) <D1>_(non) <D1> <D2> <D3> <D4> <T> nonpre rewild-type 5.60 .27 .77 .71 .37 67 1.14 .27 wild-type with(+)-cocaine^(b) 7.64 .69 .88 .30 .83 61 1.15 .13 A328W/Y332A 7.11 .87.30 .14 .01 51 1.58 1.65 A328W/Y332G 7.06 .96 .28 .52 .42 60 1.20 .35A328W/Y332A/Y419S 5.18 .84 .64 .56 .97 64 2.66 .62 ^(a)Refers to(−)-cocaine binding with wild-type human BChE or (−)-cocaine bindingwith a mutant BChE, unless indicated otherwise. ^(b)Refers to(+)-cocaine binding with wild-type human BChE. ^(c)<D1>_(non) and <D1>represent the average distances between the S198 O^(?) atom and thecarbonyl carbon of the cocaine benzoyl ester in thesimulated non-prereactive and prereactive BChE-cocaine complexes,respectively. <D2>, <D3>, <D4> refer to the average values of thesimulated distances from the carbonyl oxygen of the cocaine benzoylester to the NH hydrogen atoms of G116, G117, and A199 residues,respectively. <T> is the average value of the dihedral angle formed bythe S198 O^(?) atom and the plane of the carboxylate group of thecocaine benzoyl ester. See Scheme 2. ^(d)The root-mean-square deviation(RMSD) of the coordinates of backbone atoms in the simulated structurefrom those in the X-ray crystal structure of BChE. “nonpre” and “pre”refer to the non-prereactive and prereactive BChE-cocaine complexes,respectively.

(−)- and (+)-cocaine binding with wild-type BChE. FIG. 2 shows thebinding structures of the simulated non-prereactive and prereactivecomplexes of wild-type BChE binding with the two enantiomers of cocaine.In the non-prereactive complexes with (−)- and (+)-cocaine, the methylester group of cocaine is positioned at the top of the H438 backbone,while the cocaine benzoyl ester moiety is quasi-parallel to the C—O^(?)side chain of S198 with a dihedral angle T of −8° and 140°,respectively.

Scheme 2. Hydrolysis of (−)-cocaine and (+)-cocaine.

Here, T refers to the dihedral angle formed by S198 O^(?) and the planeof carboxylate group of the cocaine benzoyl ester as shown in thestructure diagram below.

The simulated internuclear distances between the carbonyl oxygen ofcocaine benzoyl ester group and the NH hydrogen of G116, G117, and A199are comparable for the two enantiomers. The simulated average distancesbetween the carbonyl carbon of the benzoyl ester and S198 O^(?) are 5.60Å and 5.18 Å for (−)- and (+)-cocaine, respectively. Comparing thesimulated protein backbone structures to the X-ray crystal structure ofNicolet et al, one can see from FIG. 1 that the RMSD values are allsmaller than 1.3 Å for the whole protein structures.

The MD simulations of the prereactive complexes reveal that wild-typeBChE binding with (−)-cocaine is essentially the same as the bindingwith (+)-cocaine in the binding site, except for the different positionsof methyl ester group of the substrates. The simulated average distancesbetween the carbonyl carbon of the benzoyl ester and S198 O^(?) are 3.27and 3.69 Å for (−)-cocaine and (+)-cocaine, respectively. Moreover, the(+)-cocaine is stabilized more effectively by the formation of stronghydrogen bonds with the backbone NH of residues G116, G117, and A199 assummarized above in Table 2. The cocaine benzoyl ester moiety ispositioned quasi-perpendicular to S198 C—O^(?) with a dihedral angle Tof ˜67° and ˜61° for (−)- and (+)-cocaine, respectively.

A comparison was made between the currently simulated structures of theBChE-cocaine binding with those simulated previously by using a homologymodel of BChE and it was noted that two major differences between thetwo sets of structures. By using the X-ray crystal structure inaccordance with Nicolet et al, the acyl loop is positioned on the top ofthe cocaine benzoyl ester moiety of the cocaine, whereas the acyl loopis far from the cocaine benzoyl ester moiety in the structure simulatedstarting from the homology model of Zhan et al. The RMSD of thecoordinates of backbone atoms in the previously simulated prereactiveBChE-(−)-cocaine complex from those in the X-ray crystal structure ofBChE is ˜2.0 Å for the entire protein and ˜3.0 Å for the acyl loop. TheRMSD value became ˜2.4 Å for the entire protein and ˜3.3 Å for the acylloop, when the X-ray crystal structure was replaced by the MD-simulatedprereactive BChE-(−)-cocaine complex starting from the X-ray crystalstructure. Despite these structural differences, the benzoyl ester groupof the ligand is still close to the key residues (S197, G116, and G117)in the BChE binding site. Some significant differences are associatedwith the distances between the S198 O^(?) atom and the carbonyl carbonof the cocaine benzoyl ester in non-prereactive complexes. The averagevalues of this distance in the non-prereactive complexes were ˜9.5 and˜8.5 Å for (−)- and (+)-cocaine, respectively, when a homology model wasused. Using the X-ray crystal structure to conduct the analysis,corresponding average values became ˜5.6 and ˜5.2 Å, respectively.Therefore, both (−)- and (+)-cocaine became closer to the binding sitewhen the homology model was replaced by the X-ray crystal structure.However, no significant changes of the binding in the prereactivecomplexes were observed when the used homology model was replaced by theX-ray crystal structure. The average values of the simulated distancebetween the S198 O^(?) atom and the carbonyl carbon of the cocainebenzoyl ester in the prereactive complexes are always close to ˜3.5 Åfor both (−)- and (+)-cocaine no matter whether the X-ray crystalstructure or homology model of BChE was used as the starting structure.The similar computational results obtained from the use of the X-raycrystal structure and homology model of BChE provides evidence that thefundamental structural and mechanistic insights obtained from theprevious computational studies of Zhan et al are reliable, despites theprevious simulations were performed by using the homology model when theX-ray crystal structure was not available.

Further, the simulated structures of the non-prereactive BChE-cocainecomplexes were superimposed with the corresponding prereactivecomplexes. As shown in FIG. 3, the (−)-cocaine rotation in the BChEactive site from the non-prereactive complex to the prereactive complexis hindered by some residues as the positions of Y332, A328, and F329residues in the non-prereactive complex are significantly different fromthe corresponding positions in the prereactive complex, whereas none ofthese residues hinders the (+)-cocaine rotation in the BChE active sitefrom the non-prereactive complex to the prereactive complex becausethese residues stay in nearly the same positions in the twoBChE-(+)-cocaine complexes.

(−)-cocaine binding with BChE mutants. Now that the (−)-cocaine rotationfrom the non-prereactive complex to the prereactive complex has beenknown to be the rate-determining step of the BChE-catalyzed hydrolysisof (−)-cocaine as shown by Zhan et al, useful BChE mutants should bedesigned to specifically accelerate the change from the non-prereactiveBChE-(−)-cocaine complex to the prereactive complex. The question iswhether MD simulation can be performed to help design BChE mutants thathave higher catalytic activity for (−)-cocaine hydrolysis.

In the simulated non-prereactive complex, the average distance betweenthe carbonyl carbon of cocaine benzoyl ester and S198 O^(?) is 7.6 Å forA328W/Y332A BChE and 7.1 Å for A328W/Y332G BChE, as seen in Table 2above. In the simulated prereactive complex, the average values of thisimportant internuclear distance become 3.87 and 3.96 Å for A328W/Y332Aand A328W/Y332G BChE's, respectively. Compared to the simulatedwild-type BChE-(−)-cocaine prereactive complex, the average distancesbetween the carbonyl carbon of the cocaine benzoyl ester and S198 O^(?)in the prereactive complex of (−)-cocaine with A328W/Y332A andA328W/Y332G BChE's are all slightly longer, whereas the averagedistances between the carbonyl oxygen of the cocaine benzoyl ester andthe NH of G116, G117, and A199 residues are all shorter. This providesevidence that (−)-cocaine more strongly bind with A328W/Y332A andA328W/Y332G BChE's in the prereactive complexes. More importantly, the(−)-cocaine rotation in the active site of A328W/Y332A and A328W/Y332GBChE's from the non-prereactive complex to the prereactive complex didnot cause considerable changes of the positions of A332 (or G332), W328,and F329 residues as seen in FIG. 4, compared to the (−)-cocainerotation in the active site of wild-type BChE. These results provideevidence that A328W/Y332A and A328W/Y332G BChE's should be associatedwith lower energy barriers than the wild-type for the (−)-cocainerotation from the non-prereactive complex to the prereactive complex.Further, (−)-cocaine binding with A328W/Y332G BChE is very similar tothe binding with A328W/Y332A BChE, but the position change of F329residue caused by the (−)-cocaine rotation was significant only inA328W/Y332A BChE, thus suggesting that the energy barrier for the(−)-cocaine rotation in A328W/Y332G BChE should be slightly lower thanthat in A328W/Y332A BChE.

Concerning (−)-cocaine binding with A328W/Y332A/Y419S BChE, Y419 staysdeep inside the protein and does not directly contact with the cocainemolecule. The Y419S mutation was tested because it was initiallyexpected that this mutation would further increase the free space of theactive site pocket so that the (−)-cocaine rotation could be easier.However, as seen in Table 2 above, the average distance between thecarbonyl carbon of cocaine benzoyl ester and S198 O^(?) atom in thesimulated prereactive complex was as long as 5.84 Å. The averagedistances between the carbonyl oxygen of the cocaine benzoyl ester andthe NH hydrolysis atoms of G116, G117, and A199 residues are between4.56 and 6.97 Å; no any hydrogen bond between them. In addition to theinternuclear distances, another interesting geometric parameter is thedihedral angle, T, formed by S198 O^(?) and the plane of the carboxylategroup of the cocaine benzoyl ester. As seen in Table 2, the T values inthe prereactive complexes of cocaine with wild-type BChE and all of theBChE mutants other than A328W/Y332A/Y419S BChE all slightly deviate fromthe ideal value of 900 for the nucleophilic attack of S198 O^(?) at thecarbonyl carbon of cocaine. The T value in the prereactive complex of(−)-cocaine with A328W/Y332A/Y419S BChE is 164°, which is considerablydifferent from the ideal value of 90°.

Catalytic activity. The aforementioned discussion provides evidence thatthe energy barriers for the (−)-cocaine rotation in A328W/Y332A andA328W/Y332G BChE's from the non-prereactive complex to the prereactivecomplex, the rate-determining step for the BChE-catalyzed hydrolysis of(−)-cocaine, should be lower than that in the wild-type BChE. Thus, theMD simulations predict that both A328W/Y332A and A328W/Y332G BChE'sshould have a higher catalytic activity than the wild-type BChE for(−)-cocaine hydrolysis. Further, the MD simulations also suggest thatthe energy barrier for the (−)-cocaine rotation in A328W/Y332G BChEshould be slightly lower than that in A328W/Y332A BChE and, therefore,the catalytic activity of A328W/Y332G BChE for the (−)-cocainehydrolysis should be slightly higher than the activity of A328W/Y332ABChE. In addition, the MD simulations predict that A328W/Y332A/Y419SBChE should have no catalytic activity, or have a considerably lowercatalytic activity than the wild-type, for (−)-cocaine hydrolysisbecause (−)-cocaine binds with the mutant BChE in a way that is notsuitable for the catalysis.

The catalytic efficiency (k_(cat)/K_(M)) of A328W/Y332A BChE for(−)-cocaine hydrolysis was reported to be 8.56×10⁶ M min⁻¹, which is9.39 times of the k_(cat)/K_(M) value (9.11×10⁵ M min¹) of the wild-typeBChE. To examine these theoretical predictions of the relative activityfor A328W/Y332G and A328W/Y332A/Y419S BChE's, a A328W/Y332A,A328W/Y332G, and A328W/Y332A/Y419S BChE was produced throughsite-directed mutagenesis. To minimize the possible systematicexperimental errors of the kinetic data, kinetic studies were performedwith all of three mutants under the same condition and compared thecatalytic efficiency of the A328W/Y332G and A328W/Y332A/Y419S to that ofthe A328W/Y332A for (−)-cocaine hydrolysis at benzoyl ester group. Basedon the kinetic analysis of the measured time-dependent radiometric data,the ratio of the k_(cat)/K_(M) value of A328W/Y332G BChE to thek_(cat)/K_(M) value of A328W/Y332A BChE for the (−)-cocaine hydrolysiswas determined to be ˜2.08, or A328W/Y332G BChE has a k_(cat)/K_(M)value of ˜1.78×10⁷ M min⁻¹ for the (−)-cocaine hydrolysis. Theradiometric data show no significant catalytic activity forA328W/Y332A/Y419S BChE. These experimental data are consistent with thetheoretical predictions based on the MD simulations.

Conclusion. Molecular modeling, molecular docking, and moleculardynamics (MD) simulations were performed to study cocaine binding withhuman butyrylcholinesterase (BChE) and its mutants, based on a recentlyreported X-ray crystal structure of human BChE. The MD simulations ofcocaine binding with wild-type BChE led to average BChE-cocaine bindingstructures similar to those obtained recently from the MD simulationsbased on a homology model of BChE, despite the significant differencefound at the acyl binding pocket. This confirms the fundamentalstructural and mechanistic insights obtained from the priorcomputational studies of Zhan et al based on a homology model of BChE,e.g., the rate-determining step for BChE-catalyzed hydrolysis ofbiologically active (−)-cocaine is the (−)-cocaine rotation in the BChEactive site from the non-prereactive BChE-(−)-cocaine complex to theprereactive complex.

The MD simulations further reveal that the (−)-cocaine rotation in theactive site of wild-type BChE from the non-prereactive complex to theprereactive complex is hindered by some residues such that the positionsof Y332, A328, and F329 residues in the non-prereactive complex aresignificantly different from those in the prereactive complex. Comparedto (−)-cocaine binding with wild-type BChE, (−)-cocaine more stronglybind with A328W/Y332A and A328W/Y332G BChE's in the prereactivecomplexes. More importantly, the (−)-cocaine rotation in the active siteof A328W/Y332A and A328W/Y332G BChE's from the non-prereactive complexto the prereactive complex did not cause considerable changes of thepositions of A332 or G332, W328, and F329 residues. These resultsprovide evidence that A328W/Y332A and A328W/Y332G BChE's are associatedwith lower energy barriers than wild-type BChE for the (−)-cocainerotation from the non-prereactive complex to the prereactive complex.Further, (−)-cocaine binding with A328W/Y332G BChE is very similar tothe binding with A328W/Y332A BChE, but the position change of F329residue caused by the (−)-cocaine rotation was significant only inA328W/Y332A BChE, thus suggesting that the energy barrier for(−)-cocaine rotation in A328W/Y332G BChE should be slightly lower thanthat in A328W/Y332A BChE. It has also been demonstrated that (−)-cocainebinds with A328W/Y332A/Y419S BChE in a way that is not suitable for thecatalysis.

Based on the computational results, both A328W/Y332A and A328W/Y332GBChE's have catalytic activity for (−)-cocaine hydrolysis higher thanthat of wild-type BChE and the activity of A328W/Y332G BChE should beslightly higher than that of A328W/Y332A BChE, whereas A328W/Y332A/Y419SBChE is expected to lose the catalytic activity. The computationalpredictions are completely consistent with the experimental kineticdata, providing evidence that the used computational protocol, includingmolecular modeling, molecular docking, and MD simulations, is reliablein prediction of the catalytic activity of BChE mutants for (−)-cocainehydrolysis.

Experiment 2 MD Simulations and Quantum Mechanical/Molecular Mechanical(QM/MM) Calculations Relating to a 199S/A328W/Y332G Mutant (Mutant 1)(SEQ ID NO: 1).

Generally speaking, for rational design of a mutant enzyme with a highercatalytic activity for a given substrate, one needs to design a mutationthat can accelerate the rate-determining step of the entire catalyticreaction process while the other steps are not slowed down by themutation. Reported computational modeling and experimental dataindicated that the formation of the prereactive BChE-(−)-cocaine complex(ES) is the rate-determining step of (−)-cocaine hydrolysis catalyzed bywild-type BChE as disclosed by Sun et al, Zhan et al and Hamza, A.; Cho,H.; Tai, H.-H.; Zhan, C.-G. J. Phys. Chem. B 2005, 109, 4776, hereinincorporated by reference, whereas the rate-determining step of thecorresponding (+)-cocaine hydrolysis is the chemical reaction processconsisting of four individual reaction steps disclosed by Zhan et al andshown in Scheme 3 and Scheme 4 below.

Scheme 3. Schematic representation of BChE-catalyzed hydrolysis of(−)-cocaine. Only QM-treated high-layer part of the reaction system inthe QM/MM calculations are drawn. Notation [H] refers to a non-hydrogenatom in the MM-treated low-layer part of the protein and the cutcovalent bond with this atom is saturated by a hydrogen atom. The dashlines in the transition state structures represent the transition bonds.

This mechanistic understanding is consistent with the experimentalobservation of Sun et al, that the catalytic rate constant of wild-typeBChE against (+)-cocaine is pH-dependent, whereas that of the sameenzyme against (−)-cocaine is independent of the pH. The pH-dependenceof the rate constant for (+)-cocaine hydrolysis is clearly associatedwith the protonation of H438 residue in the catalytic triad (S198, H438,and E325). For the first and third steps of the reaction process, whenH438 is protonated, the catalytic triad cannot function and, therefore,the enzyme becomes inactive. The lower the pH of the reaction solutionis, the higher the concentration of the protonated H438 is, and thelower the concentration of the active enzyme is. Hence, the rateconstant was found to decrease with decreasing the pH of the reactionsolution for the enzymatic hydrolysis of (+)-cocaine.

Based on the above mechanistic understanding, the efforts for rationaldesign of the BChE mutants reported in literature have been focused onhow to improve the ES formation process. Indeed, several BChE mutants,including A328W, A328W/Y332A, A328W/Y332G, and F227A/S287G/A328W/Y332M,have been found to have a significantly higher catalytic efficiency(k_(cat)/K_(M)) against (−)-cocaine; these mutants of BChE have anapproximate 9 to 34-fold improved catalytic efficiency against(−)-cocaine. Experimental observation also indicated that the catalyticrate constant of A328W/Y332A BChE is pH-dependent for both (−)- and(+)-cocaine. The pH-dependence reveals that for both (−)- and(+)-cocaine, the rate-determining step of the hydrolysis catalyzed byA328W/Y332A BChE should be either the first or the third step of thereaction process. Further, if the third step were rate determining, thenthe catalytic efficiency of the A328W/Y332A mutant against (−)-cocaineshould be as high as that of the same mutant against (+)-cocaine becausethe (−)- and (+)-cocaine hydrolyses share the same third and fourthsteps (see Scheme 3). However, it has also been observed that theA328W/Y332A mutant only has a ˜9-fold improved catalytic efficiencyagainst (−)-cocaine, whereas the A328W/Y332A mutation does not changethe high catalytic activity against (+)-cocaine. This analysis of theexperimental and computational data available in literature clearlyshows that the rate-determining step of (−)-cocaine hydrolysis catalyzedby the A328W/Y332A mutant should be the first step of the chemicalreaction process. Further, recently reported computational modeling alsosuggests that the formation of the prereactive BChE-(−)-cocaine complex(ES) is hindered mainly by the bulky side chain of Y332 residue inwild-type BChE, but the hindering can be removed by the Y332A or Y332Gmutation. Therefore, starting from the A328W/Y332A or A328W/Y332Gmutant, the truly rational design of further mutation(s) to improve thecatalytic efficiency of BChE against (−)-cocaine should aim to decreasethe energy barrier for the first reaction step without significantlyaffecting the ES formation and other chemical reaction steps.

The following rational design of a high-activity mutant of BChE against(−)-cocaine is based on detailed computational modeling of thetransition state for the rate-determining step (i.e., the first step ofthe chemical reaction process). Molecular dynamics (MD) simulations andhybrid quantum mechanical/molecular mechanical (QM/MM) calculations wereperformed to model the protein environmental effects on thestabilization of the transition-state structure for BChE-catalyzedhydrolysis of (−)-cocaine. The simulated and calculated results indicatethat the transition-state structure can be stabilized better by theprotein environment in A199S/A328W/Y332G mutant of BChE than that in thewild-type. The computational modeling led to a prediction of the highercatalytic efficiency for the A199S/A328W/Y332G mutant against(−)-cocaine. The prediction has been confirmed by wet experimental testsshowing that the A199S/A328W/Y332G mutant has a significantly improvedcatalytic efficiency against (−)-cocaine. All of the obtained resultsclearly demonstrate that directly modeling the transition-statestructure provides a reliable computational approach to the rationaldesign of a high-activity mutant of BChE against (−)-cocaine.

MD simulations. It should be stressed that a critical issue exists withregard to any MD simulation on a transition state. In principle, MDsimulation using a classical force field (molecular mechanics) can onlysimulate a stable structure corresponding to a local minimum on thepotential energy surface, whereas a transition state during a reactionprocess is always associated with a first-order saddle point on thepotential energy surface. Hence, MD simulation using a classical forcefield cannot directly simulate a transition state without any restrainton the geometry of the transition state. Nevertheless, if one cantechnically remove the freedom of imaginary vibration in the transitionstate structure, then the number of vibrational freedoms (normalvibration modes) for a nonlinear molecule will decrease from 3N-6. Thetransition state structure is associated with a local minimum on thepotential energy surface within a subspace of the reduced vibrationalfreedoms, although it is associated with a first-order saddle point onthe potential energy surface with all of the 3N-6 vibrational freedoms.Theoretically, the vibrational freedom associated with the imaginaryvibrational frequency in the transition state structure can be removedby appropriately freezing the reaction coordinate. The reactioncoordinate corresponding to the imaginary vibration of the transitionstate is generally characterized by a combination of some key geometricparameters. These key geometric parameters are bond lengths of theforming and breaking covalent bonds for BChE-catalyzed hydrolysis ofcocaine, as seen in Scheme 3. Thus, one just needs to maintain the bondlengths of the forming and breaking covalent bonds during the MDsimulation on a transition state. Technically, one can maintain the bondlengths of the forming and breaking covalent bonds by simply fixing allatoms within the reaction center, by using some constraints on theforming and breaking covalent bonds, or by redefining the forming andbreaking covalent bonds. It should be pointed out that the only purposeof performing such type of MD simulation on a transition state is toexamine the dynamic change of the protein environment surrounding thereaction center and the interaction between the reaction center and theprotein environment. For this study, of interest is the simulatedstructures, as the total energies calculated in this way aremeaningless.

The initial BChE structures used in the MD simulations were preparedbased on the previous MD simulation in accordance with Hamza et al onthe prereactive ES complex for wild-type BChE with (−)-cocaine in waterby using Amber7 program package. The previous MD simulations on theprereactive BChE-(−)-cocaine complex (ES) started from the X-ray crystalstructure of Nicolet et al deposited in the Protein Data Bank (pdb code:1 POP). The present MD simulation on the transition state for the firststep (TS1) was performed in such a way that bond lengths of thepartially formed and partially broken covalent bonds in the transitionstate were all constrained to be the same as those obtained from ourprevious ab initio reaction coordinate calculations on the modelreaction system of wild-type BChE in accordance with Zhan et al. Thepartially formed and partially broken covalent bonds in the transitionstate will be called “transition” bonds below, for convenience. Asufficiently long MD simulation with the transition bonds constrainedshould lead to a reasonable protein environment stabilizing the reactioncenter in the transition-state structure simulated. Further, thesimulated TS1 structure for wild-type BChE with (−)-cocaine was used tobuild the initial structures of TS1 for the examined BChE mutants with(−)-cocaine; only the side chains of mutated residues needed to bechanged.

The partial atomic charges for the non-standard residue atoms, includingcocaine atoms, in the TS1 structures were calculated by using the RESPprotocol implemented in the Antechamber module of the Amber7 packagefollowing electrostatic potential (ESP) calculations at ab initioHF/6-31G* level using Gaussian03 program known in the art. Thegeometries used in the ESP calculations came from those obtained fromthe previous ab initio reaction coordinate calculations of Zhan et al,but the functional groups representing the oxyanion hole were removed.Thus, residues G116, G117, and A199 were the standard residues assupplied by Amber7 in the MD simulations. The general procedure forcarrying out the MD simulations in water is essentially the same as thatused in our previously reported other computational studies includingZhan et al, Hamza et al and (a) Zhan, C.-G.; Norberto de Souza, O.;Rittenhouse, R.; Ornstein, R. L. J. Am. Chem. Soc. 1999, 121, 7279, (b)Koca, J.; Zhan, C.-G.; Rittenhouse, R.; Ornstein, R. L. J. Am. Chem.Soc. 2001, 123, 817, (c) Koca, J.; Zhan, C.-G.; Rittenhouse, R. C.;Ornstein, R. L. J. Comput. Chem. 2003, 24, 368, (d) Hamza, A.; Cho, H.;Tai, H.-H.; Zhan, C.-G. Bioorg. Med. Chem. 2005, 13, 4544, herein allincorporated by reference. Each aforementioned starting TS1 structurewas neutralized by adding chloride counterions and was solvated in arectangular box of TIP3P water molecules with a minimum solute-walldistance of 10 Å as described by Jorgensen, W. L.; Chandrasekhar, J.;Madura, J.; Klein, M. L. J. Chem. Phys. 1983, 79, 926, hereinincorporated by reference. The total numbers of atoms in the solvatedprotein structures for the MD simulations are nearly 70,000, althoughthe total number of atoms of BChE and (−)-cocaine is only 8417 (for thewild-type BChE). All of the MD simulations were performed by using theSander module of Amber7 package. The solvated systems were carefullyequilibrated and fully energy minimized. These systems were graduallyheated from T=10 K to T=298.15 K in 30 ps before running the MDsimulation at T=298.15 K for 1 ns or longer, making sure that a stableMD trajectory was obtained for each of the simulated TS1 structures. Thetime step used for the MD simulations was 2 fs. Periodic boundaryconditions in the NPT ensemble at T=298.15 K with Berendsen temperaturecoupling and P=1 atm with isotropic molecule-based scaling were applied.The SHAKE algorithm was used to fix all covalent bonds containinghydrogen atoms. The non-bonded pair list was updated every 10 steps. Theparticle mesh Ewald (PME) method in accordance with Essmann, U.; Perera,L.; Berkowitz, M. L.; Darden, T. A.; Lee, H., Pedersen, L. G. J. Chem.Phys. 1995, 98, 10089, herein incorporated by reference, was used totreat long-range electrostatic interactions. A residue-based cutoff of10 Å was utilized to the non-covalent interactions. The coordinates ofthe simulated systems were collected every 1 ps during the production MDstages.

QM/MM calculations. For each TS1 structure examined, after the MDsimulation was completed and a stable MD trajectory was obtained, all ofthe collected snapshots of the simulated structure, excluding thosebefore the trajectory was stabilized, were averaged and furtherenergy-minimized. The energy-minimized average structure (with thetransition bonds constrained) was used as an initial geometry to carryout a further geometry optimization by using the ONIOM approach ofDapprich, S.; Komaromi, I.; Byun, K. S.; Morokuma, K.; Frisch, M. J. J.Mol. Struct. (Theochem) 1999, 461, 1-21, herein incorporated byreference, implemented in the Gaussian03 program of Frisch, M. J. et alGaussian 03, Revision A.1, Gaussian, Inc., Pittsburgh, Pa., 2003, hereinincorporated by reference. Two layers were defined in the present ONIOMcalculation: the high layer, as depicted in Scheme 3 above, wascalculated quantum mechanically at the ab initio HF/3-21G level, whereasthe low layer was calculated molecular mechanically by using the Amberforce field as used in our MD simulation and energy minimization withthe Amber7 program. The ONIOM calculations at the HF/3-21 G: Amber levelin this study are a type of QM/MM calculations of Vreven, T.; Morokuma,K. J. Chem. Phys. 2000, 113, 2969-2975 and Frisch, M.; Vreven, T.;Schlegel, H. B.; Morokuma, K. J. Comput. Chem. 2003, 24, 760-769, hereinboth incorporated by reference. Previous reaction coordinatecalculations with an active site model of wild-type BChE demonstratethat the HF/3-21G level is adequate for the geometry optimization ofthis enzymatic reaction system shown by Zhan et al, although the finalenergy calculations for calculating the energy barriers must be carriedout at a higher level. As depicted in Scheme 3, for all of these QM/MMcalculations, the same part of the enzyme was included in the QM-treatedhigh layer. So, the QM-treated high layer included (−)-cocaine, keyfunctional groups from the catalytic triad (S198, H438, and E325), andthe three residues, i.e., G116, G117, and A199 (or S199 for a particularmutant, see Scheme 4), of the possible oxyanion hole, whereas the entireenzyme structure of BChE was included in the MM-treated low layer. Alanguage computer program was developed to automatically generate theinput files for the ONIOM calculations following the MD simulations andsubsequent energy minimizations in order to make sure that the atomtypes used for all low-layer atoms are the same as what were used in theAmber7. During the TS1 geometry optimization using the two-layer ONIOM,the length of a key transition C—O bond was fixed which dominates thereaction coordinate; all of the other transition bond lengths wererelaxed. The C and O atoms in the key transition C—O bond are thecarbonyl carbon of (−)-cocaine benzoyl ester and the O^(γ) atom of S198,respectively, according to the previous reaction coordinate calculationswith an active site model of wild-type BChE of Zhan et al.

Most of the MD simulations and QM/MM calculations were performed inparallel on an HP supercomputer (Superdome, with 256 shared-memoryprocessors) at the Center for Computational Sciences, University ofKentucky. Some of the computations were carried out on a 34-processorsIBM x335 Linux cluster and SGI Fuel workstations.

Scheme 4. Schematic representation of the first reaction step for(−)-cocaine hydrolysis catalyzed by a BChE mutant with an A199Smutation.

Experimental materials. Cloned pfu DNA polymerase and Dpn I endonucleasewere obtained from Stratagene (La Jolla, Calif.). ³H-(−)-cocaine (50Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, Mass.).The expression plasmid pRc/CMV was a gift from Dr. O. Lockridge,University of Nebraska Medical Center (Omaha, Nebr.). Alloligonucleotides were synthesized by the Integrated DNA Technologies,Inc. The QIAprep Spin Plasmid Miniprep Kit and Qiagen plasmidpurification kit and QIAquick PCR purification kit were obtained fromQiagen (Santa Clarita, Calif.). Human embryonic kidney 293T/17 cellswere from ATCC (Manassas, Va.). Dulbecco's modified Eagle's medium(DMEM) was purchased from Fisher Scientific (Fairlawn, N.J.).Oligonucleotide primers were synthesized by the Integrated DNATechnologies and Analysis Facility of the University of Kentucky. 3, 3′,5,5′-Tetramethylbenzidine (TMB) was obtained from Sigma (Saint Louis,Mo.). Anti-butyrylcholinesterase (mouse monoclonal antibody, Product #HAH002-01) was purchased from AntibodyShop (Gentofte, Denmark) and Goatanti-mouse IgG HRP conjugate from Zymed (San Francisco, Calif.).

Site-directed mutagenesis, protein expression, and BChE activity assay.Site-directed mutagenesis of human BChE cDNA was performed by using theQuikChange method of Braman et al. Mutations were generated fromwild-type human BChE in a pRc/CMV expression plasmid in accordance with(a) Masson, P.; Legrand, P.; Bartels, C. F.; Froment, M.-T.; Schopfer,L. M.; Lockridge, O. Biochemistry 1997, 36, 2266, (b) Masson, P.; Xie,W., Froment, M-T.; Levitsky, V.; Fortier, P.-L.; Albaret, C.; Lockridge,O. Biochim. Biophys. Acta 1999, 1433, 281, (c) Xie, W.; Altamirano, C.V.; Bartels, C. F.; Speirs, R. J.; Cashman, J. R.; Lockridge, O. Mol.Pharmacol. 1999, 55, 83, (d) Duysen, E. G.; Bartels, C. F.; Lockridge,O. J. Pharmacol. Exp. Ther. 2002, 302, 751, (e) Nachon, F.; Nicolet, Y.;Viguie, N.; Masson, P.; Fontecilla-Camps, J. C.; Lockridge, O. Eur. J.Biochem. 2002, 269, 630, herein all incorporated by reference. Usingplasmid DNA as template and primers with specific base-pair alterations,mutations were made by polymerase chain reaction with Pfu DNApolymerase, for replication fidelity. The PCR product was treated withDpn I endonuclease to digest the parental DNA template. Modified plasmidDNA was transformed into Escherichia coli, amplified, and purified. TheDNA sequences of the mutants were confirmed by DNA sequencing. BChEmutants were expressed in human embryonic kidney cell line 293T/17.Cells were grown to 80-90% confluence in 6-well dishes and thentransfected by Lipofectamine 2000 complexes of 4 μg plasmid DNA per eachwell. Cells were incubated at 37° C. in a CO₂ incubator for 24 hours andcells were moved to 60-mm culture vessel and cultured for four moredays. The culture medium [10% fetal bovine serum in Dulbecco's modifiedEagle's medium (DMEM)] was harvested for a BChE activity assay. Tomeasure (−)-cocaine and benzoic acid, the product of (−)-cocainehydrolysis catalyzed by BChE, sensitive radiometric assays were usedbased on toluene extraction of [³H]-(−)-cocaine labeled on its benzenering in accordance with Sun et al. In brief, to initiate the enzymaticreaction, 100 nCi of [³H]-(−)-cocaine was mixed with 100 μl of culturemedium. The enzymatic reactions proceeded at room temperature (25° C.)for varying time. The reactions were stopped by adding 300 μl of 0.02 MHCl, which neutralized the liberated benzoic acid while ensuring apositive charge on the residual (−)-cocaine. [³H]benzoic acid wasextracted by 1 ml of toluene and measured by scintillation counting.Finally, the measured time-dependent radiometric data were fitted to thekinetic equation so that the catalytic efficiency (k_(cat)/K_(M)) wasdetermined along with the use of an enzyme-linked immunosorbent assay(ELISA) described below.

Enzyme-linked immunosorbent assay (ELISA). The ELISA buffers used in thepresent study are the same as those described in the literature such as(a) Brock, A.; Mortensen, V.; Loft, A. G. R.; Nergaard-Pedersen, B. J.Clin. Chem. Clin. Biochem. 1990, 28, 221-224, (b) Khattab, A. D.;Walker, C. H.; Johnston, G.; Siddiqui, M. K. Saphier, P. W.Environmental Toxicology and Chemistry 1994, 13, 1661-1667, herein bothincorporated by reference. The coating buffer was 0.1 M sodiumcarbonate/bicarbonate buffer, pH 9.5. The diluent buffer (EIA buffer)was potassium phosphate monobasic/potassium phosphate monohydratebuffer, pH 7.5, containing 0.9% sodium chloride and 0.1% bovine serumalbumin. The washing buffer (PBS-T) was 0.01 M potassium phosphatemonobasic/potassium phosphate monohydrate buffer, pH 7.5, containing0.05% (v/v) Tween-20. All the assays were performed in triplicate. Eachwell of an ELISA microtiter plate was filled with 100 μl of the mixturebuffer consisting of 20 μl culture medium and 80 μl coating buffer. Theplate was covered and incubated overnight at 4° C. to allow the antigento bind to the plate. The solutions were then removed and the wells werewashed four times with PBS-T. The washed wells were filled with 200 μldiluent buffer and kept shaking for 1.5 h at room temperature (25° C.).After washing with PBS-T for four times, the wells were filled with 100μl antibody (1:8000) and were incubated for 1.5 h, followed by washingfor four times. Then, the wells were filled with 100 μl goat anti-mouseIgG HRP conjugate complex diluted to a final 1:3000 dilution, and wereincubated at room temperature for 1.5 h, followed by washing for fourtimes. The enzyme reactions were started by addition of 100 μl substrate(TMB) solution. The reactions were stopped after 15 min by the additionof 100 μl of 2 M sulfuric acid, and the absorbance was read at 460 nmusing a Bio-Rad ELISA plate reader.

Hydrogen bonding revealed by the MD simulations. In accordance with oneaspect of the present invention, namely the generation of high-activitymutants of BChE against (−)-cocaine, the present invention includespredicting some possible mutations that can lower the energy of thetransition state for the first chemical reaction step (TS1) and,therefore, lower the energy barrier for this critical reaction step.Apparently, a mutant associated with the stronger hydrogen bondingbetween the carbonyl oxygen of (−)-cocaine benzoyl ester and theoxyanion hole of the BChE mutant in the TS1 structure may potentiallyhave a more stable TS1 structure and, therefore, a higher catalyticactivity for (−)-cocaine hydrolysis. Hence, the hydrogen bonding withthe oxyanion hole in the TS1 structure is a crucial factor affecting thetransition state stabilization and the catalytic activity. The possibleeffects of some mutations on the hydrogen bonding were examined byperforming MD simulations and QM/MM calculations on the TS1 structuresfor (−)-cocaine hydrolysis catalyzed by the wild-type and variousmutants BChE's.

The MD simulation in water was performed for 1 ns or longer to make surea stable MD trajectory was obtained for each simulated TS1 structurewith the wild-type or mutant BChE. The MD trajectories actually becamestable quickly, so were the H—O distances involved in the potentialhydrogen bonds between the carbonyl oxygen of (−)-cocaine and theoxyanion hole of BChE.

Depicted in FIG. 5 are plots of four important H O distances in theMD-simulated TS1 structure versus the simulation time for (−)-cocainehydrolysis catalyzed by A328W/Y332A or A199S/A328W/Y332G BChE, alongwith the root-mean-square deviation (RMSD) of the simulated positions ofbackbone atoms from those in the corresponding initial structure. TracesD1, D2, and D3 refer to the distances between the carbonyl oxygen of(−)-cocaine and the NH hydrogen of G116, G117, and S199, respectively.Trace D4 is the internuclear distance between the carbonyl oxygen of(−)-cocaine and the hydroxyl hydrogen of the S199 side chain whichexists only in A199S/A328W/Y332G BChE. RMSD represents theroot-mean-square deviation (in A) of the simulated positions of theprotein backbone atoms from those in the initial structure. The H Odistances in the simulated TS1 structures corresponding to the wild-typeBChE and the two mutants are summarized in Table 3. The H—O distancesbetween the carbonyl oxygen of (−)-cocaine and the peptidic NH hydrogenatoms of G116, G17, and A199 (or S199) of BChE are denoted by D1, D2,and D3, respectively, in Table 2 and FIG. 1. D4 in Table 3 and FIG. 5refers to the H—O distance between the carbonyl oxygen of (−)-cocaineand the hydroxyl hydrogen of S199 side chain in the simulated TS1structure corresponding to the A199S/A328W/Y332G mutant, mutant (1) (SEQID NO: 2).

TABLE 3 Summary of the MD-simulated and QM/MM-optimized key distances(in Å) and the calculated total hydrogen-bonding energies (HBE, inkcal/mol) between the oxyanion hole and the carbonyl oxygen of(−)-cocaine benzoyl ester in the first transition state (TS1).Distance^(a) Transition State Method D1 D2 D3 D4 Total HBE^(b) TS1 for(−)-cocaine MD Average 4.59 2.91 1.92 −5.5 (−4.6) hydrolysis catalyzedMaximum 5.73 4.14 2.35 by wild-type BChE Minimum 3.35 1.97 1.61Fluctuation 0.35 0.35 0.12 QM/MM 4.10 2.21 2.05 −4.2 TS1 for (−)-cocaineMD Average 3.62 2.35 1.95 −6.2 (−4.9) hydrolysis catalyzed Maximum 4.353.37 3.02 by A328W/Y332A Minimum 2.92 1.78 1.61 mutant of BChEFluctuation 0.23 0.27 0.17 QM/MM 3.39 2.05 2.47 −3.3 TS1 for (−)-cocaineMD Average 5.30 2.21 1.94 2.15 −9.7 (−7.4) hydrolysis catalyzed Maximum6.08 3.06 2.47 3.27 by A199S/A328W/ Minimum 4.57 1.71 1.66 1.56 Y332Gmutant of Fluctuation 0.22 0.20 0.13 0.29 BChE QM/MM 4.83 2.09 1.91 2.59−7.46 ^(a)D1, D2, and D3 represent the internuclear distances betweenthe carbonyl oxygen of cocaine benzoyl ester and the NH hydrogen ofresidues #116 (i.e., G116), #117 (i.e., G117), and #199 (i.e., A199 orS199) of BChE, respectively. D4 is the internuclear distance between thecarbonyl oxygen of cocaine benzoyl ester and the hydroxyl hydrogen ofS199 side chain in the A199S/A328W/Y332G mutant. ^(b)The total HBE valueunder MD is the average of the HBE values calculated by using theinstantaneous distances in all of the snapshots. The valuein parenthesis is the total HBE value calculated by using theMD-simulated average distances. The total HBE value under QM/MM wasevaluated by using the QM/MM-optimized distances.

As seen in Table 3, the simulated H—O distance D1 is always too long forthe peptidic NH of G116 to form a N—H—O hydrogen bond with the carbonyloxygen of (−)-cocaine. In the simulated TS1 structure corresponding towild-type BChE, the carbonyl oxygen of (−)-cocaine formed a firm N—H—Ohydrogen bond with the peptidic NH hydrogen atom of A199 residue; thesimulated H O distance was 1.61 to 2.35 Å, with an average value of 1.92Å. Meanwhile, the carbonyl oxygen of (−)-cocaine also had a partialN—H—O hydrogen bond with the peptidic NH hydrogen atom of G117 residue;the simulated H O distance was 1.97 to 4.14 Å (the average value: 2.91Å). In the simulated TS1 structure corresponding to the A328W/Y332Amutant, the simulated average H—O distances with the peptidic NHhydrogen of G117 and A199 are 2.35 and 1.95 Å, respectively. Thesedistances suggest a slightly weaker N—H—O hydrogen bond with A199, but astronger N—H—O hydrogen bond with G117, in the simulated TS1 structurecorresponding to the A328W/Y332A mutant. The overall strength of thehydrogen bonding between the carbonyl oxygen of (−)-cocaine and theoxyanion hole of the enzyme is not expected to change considerably whenwild-type BChE is replaced by the A328W/Y332A mutant.

However, the story for the simulated TS1 structure associated with theA199S/A328W/Y332G mutant was remarkably different. As one can see fromScheme 4, FIG. 5, and Table 3, when residue #199 becomes a serine (i.e.,S199), the hydroxyl group on the side chain of S199 can alsohydrogen-bond to the carbonyl oxygen of (−)-cocaine to form an O—H—Ohydrogen bond, in addition to the two N—H—O hydrogen bonds with thepeptidic NH of G117 and S199. The simulated average H—O distances withthe peptidic NH hydrogen of G117, peptidic NH hydrogen of S199, andhydroxyl hydrogen of S199 are 2.21, 1.94, and 2.15 Å, respectively. Dueto the additional O—H—O hydrogen bond, the overall strength of thehydrogen bonding with the modified oxyanion hole of A199S/A328W/Y332GBChE should be significantly stronger than that of the wild-type andA328W/Y332A BChE's.

To better represent the overall strength of hydrogen bonding between thecarbonyl oxygen of (−)-cocaine and the oxyanion hole in a MD-simulatedTS1 structure, the hydrogen bonding energy (HBE) associated with eachsimulated H—O distance was estimated by using the empirical HBE equationimplemented in AutoDock 3.0 program suite in accordance with (a) Morris,G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew.R. K.; Olson, A. J. J. Comput. Chem. 1998, 19, 1639-1662, and based onthe general HBE equation, HBE(r)≈5εr₀ ¹²/r¹²−6εr₀ ¹⁰/r¹⁰, in which r isthe H—O distance in the considered hydrogen bond and r₀ is the minimumvalue of the H—O distance for which the HBE equation can be used. Forthe calculation, r₀=1.52 Å, because it is the shortest H—O distancefound in all of our MD simulations. The ε value was determined by usingthe condition that HBE(r)=−5.0 kcal/mol when r=1.90 Å, hereinincorporated by reference. Specifically, for each hydrogen bond with thecarbonyl oxygen of (−)-cocaine, a HBE value can be evaluated with eachsnapshot of the MD-simulated structure. The final HBE of theMD-simulated hydrogen bond is considered to be the average HBE value ofall snapshots taken from the stable MD trajectory. The estimated totalHBE value for the hydrogen bonds between the carbonyl oxygen of(−)-cocaine and the oxyanion hole in each simulated TS1 structure isgiven in Table 3.

The HBE for each hydrogen bond was estimated by using the MD-simulatedaverage H—O distance. As seen in Table 3, the total hydrogen-bondingenergies (i.e., −4.6, −4.9, and −7.4 kcal/mol for the wild-type,A328W/Y332A, and A199S/A328W/Y332G BChE's, respectively) estimated inthis way are systematically higher (i.e., less negative) than thecorresponding total hydrogen-bonding energies (i.e., −5.5, −6.2, and−9.7 kcal/mol) estimated in the aforementioned way. However, the twosets of total HBE values are qualitatively consistent in terms of therelative hydrogen-bonding strengths in the three simulated TS1structures. In particular, the two sets of total HBE values consistentlyreveal that the overall strength of the hydrogen bonding between thecarbonyl oxygen of (−)-cocaine and the oxyanion hole in the simulatedTS1 structure for A199S/A328W/Y332G BChE is significantly higher thanthat for the wild-type and A328W/Y332A BChE's.

Hydrogen bonding based on the QM/MM calculations. The above conclusionobtained from the MD simulations was further examined by carrying outQM/MM calculations. Although this enzymatic reaction system is too largeto calculate the QM/MM force constant matrix required in the automatedsearch for a first-order saddle point corresponding to TS1, a partialgeometry optimization was performed by fixing the length of thetransition C—O bond between the carbonyl carbon of (−)-cocaine and the Oatom of S198 in the QM/MM calculation. This transition C—O bond lengthdominates the reaction coordinate, according to the previousfirst-principle reaction coordinate calculations, in accordance withZhan et al, with an active model of wild-type BChE. In the partialgeometry optimization, the transition C—O bond length was fixed at thatin the TS1 geometry optimized previously by performing thefirst-principle reaction coordinate calculation with an active sitemodel of wild-type BChE. This partially optimized geometry should beclose to the precisely defined TS1 geometry associated with afirst-order saddle point on the potential energy surface, particularlyfor the hydrogen bonding between the carbonyl oxygen of (−)-cocaine andthe oxyanion hole of BChE.

The QM/MM results summarized in Table 3 demonstrate two hydrogen bondsin each of the QM/MM-optimized TS1 structures. Specifically, D2=2.21 Åand D3=2.05 Å in the optimized TS1 structure for wild-type BChE; D2=2.05Å and D3=2.47 Å in the optimized TS1 structure for the A328W/Y332Amutant; D2=2.09 Å, D3=1.91 Å, and D4=2.59 Å in the optimized TS1structure for the A199S/A328W/Y332G mutant. Although the QM/MM-optimizedindividual H—O distances and the estimated HBE values are different fromthe corresponding values for individual hydrogen bonds, the relativetotal HBE values (i.e., −4.2, −3.3, and −7.46 kcal/mol for thewild-type, A328W/Y332A, and A199S/A328W/Y332G BChE's, respectively)estimated from these optimized distances are qualitatively consistentwith the corresponding total HBE values (i.e., −4.6, −4.9, and −7.4kcal/mol) estimated from the MD-simulated average H—O distances.

It should be pointed out that the absolute HBE values estimated in thisstudy are not expected to be accurate, as different computationalapproaches led different HBE values. Nevertheless, for the purpose ofour computational design of a high-activity mutant of BChE, one onlyneeds to know the relative strength of the hydrogen bonding based on theestimated relative total HBE values of different mutants. The three setsof total HBE values (Table 3) estimated from the MD-simulated andQM/MM-optimized H—O distances all consistently demonstrate: (1) theoverall strength of hydrogen bonding between the carbonyl oxygen of(−)-cocaine and the oxyanion hole in the TS1 structure for (−)-cocainehydrolysis catalyzed by A328W/Y332A BChE should be close to that in theTS1 structure for (−)-cocaine hydrolysis catalyzed by wild-type BChE;(2) the overall hydrogen bonding between the carbonyl oxygen of(−)-cocaine and the oxyanion hole in the TS1 structure for (−)-cocainehydrolysis catalyzed by A199S/A328W/Y332G BChE should be significantlystronger than that in the TS1 structure for (−)-cocaine hydrolysiscatalyzed by A328W/Y332A BChE.

Catalytic activity. The computational results discussed above suggestthat the transition state for the first chemical reaction step (TS1) of(−)-cocaine hydrolysis catalyzed by the A199S/A328W/Y332G mutant shouldbe significantly more stable than that by the A328W/Y332A mutant, due tothe significant increase of the overall hydrogen bonding between thecarbonyl oxygen of (−)-cocaine and the oxyanion hole of the enzyme inthe TS1 structure. The aforementioned analysis of the literature, namelySun et al and Hamza et al, also indicate that the first chemicalreaction step associated with TS1 should be the rate-determining step of(−)-cocaine hydrolysis catalyzed by a BChE mutant including Y332A orY332G mutation, although the formation of the prereactiveenzyme-substrate complex (ES) is the rate-determining step for(−)-cocaine hydrolysis catalyzed by wild-type BChE. This providesevidence of a clear correlation between the TS1 stabilization and thecatalytic activity of A328W/Y332A and A199S/A328W/Y332G BChE's for(−)-cocaine hydrolysis: the more stable the TS1 structure, the lower theenergy barrier, and the higher the catalytic activity. Thus, both the MDsimulations and QM/MM calculations predict that A199S/A328W/Y332G BChEshould have a higher catalytic activity than A328W/Y332A BChE for(−)-cocaine hydrolysis.

The catalytic efficiency (k_(cat)/K_(M)) of A328W/Y332A BChE for(−)-cocaine hydrolysis was reported by Sun et al to be ˜8.6×10⁶ M min⁻¹,which is ˜9.4 times of the k_(cat)/K_(M) value (˜9.1×10⁵ M min⁻¹) ofwild-type BChE. To examine the theoretical prediction of the highercatalytic activity for A199S/A328W/Y332G BChE, A328W/Y332A andA199S/A328W/Y332G mutants of BChE were produced through site-directedmutagenesis. To minimize the possible systematic experimental errors ofthe kinetic data, kinetic studies were performed with the two mutantsand wild-type BChE under the same condition and compared the catalyticefficiency of A328W/Y332A and A199S/A328W/Y332G BChE's to that of thewild-type for (−)-cocaine hydrolysis at benzoyl ester group. Based onthe kinetic analysis of the measured time-dependent radiometric data andthe ELISA data, the ratio of the k_(cat)/K_(M) value of A328W/Y332A BChEto the k_(cat)/K_(M) value of wild-type BChE for (−)-cocaine hydrolysiswas determined to be ˜8.6. The determined catalytic efficiency ratio of˜8.6 is in good agreement with the ratio of ˜9.4 determined by Sun etal. Further, by using the same experimental protocol, the ratio of thek_(cat)/K_(M) value of A199S/A328W/Y332G BChE to the k_(cat)/K_(M) valueof A328W/Y332A BChE for (−)-cocaine hydrolysis was determined to be˜7.2. These data indicate that the ratio of the k_(cat)/K_(M) value ofA199S/A328W/Y332G BChE to the k_(cat)/K_(M) value of wild-type BChE for(−)-cocaine hydrolysis should be ˜7.2×8.6=˜62 or ˜7.2×9.4=˜68. Thus,A199S/A328W/Y332G BChE has a ˜(65±6)-fold improved catalytic efficiencyagainst (−)-cocaine compared to the wild-type, or A199S/A328W/Y332G BChEhas a k_(cat)/K_(M) value of ˜(5.9±0.5)×10⁷ M min⁻¹ for (−)-cocainehydrolysis.

Very recently reported F227A/S287G/A328W/Y332M BChE (i.e., AME-359,k_(cat)/K_(M)=3.1×10⁷ M min⁻¹) has a ˜34-fold improved catalyticefficiency for (−)-cocaine hydrolysis. AME-359 has the highest catalyticefficiency against (−)-cocaine within all of the BChE mutants reportedin literature prior to the present study. The catalytic efficiency forour A199S/A328W/Y332G BChE is about two times of that for AME-359against (−)-cocaine.

Conclusion. Molecular dynamics (MD) simulations and hybrid quantummechanical/molecular mechanical (QM/MM) calculations on the transitionstate for the first chemical reaction step (TS1) of (−)-cocainehydrolysis catalyzed by butyrylcholinesterase (BChE) mutants lead to abetter understanding of the effects of protein environment on thetransition state stabilization. All of the computational resultsconsistently demonstrate that the overall hydrogen bonding between thecarbonyl oxygen of (−)-cocaine benzoyl ester and the oxyanion hole ofBChE in the TS1 structure for (−)-cocaine hydrolysis catalyzed byA199S/A328W/Y332G BChE should be significantly stronger than that in theTS1 structure for (−)-cocaine hydrolysis catalyzed by the wild-type andA328W/Y332A BChE's. Thus, both the MD simulations and QM/MM calculationspredict that A199S/A328W/Y332G BChE should have a lower energy barrierfor the chemical reaction process and, therefore, a higher catalyticefficiency (k_(cat)/K_(M)) for (−)-cocaine hydrolysis; A328W/Y332A BChEhas been known to have a ˜9-fold improved catalytic efficiency for(−)-cocaine hydrolysis. The theoretical prediction has been confirmed bywet experimental tests showing a ˜(65±6)-fold improved catalyticefficiency for A199S/A328W/Y332G BChE against (−)-cocaine compared tothe wild-type BChE. The k_(cat)/K_(M) value determined forA199S/A328W/Y332G BChE is about two times of the k_(cat)/K_(M) value forF227A/S287G/A328W/Y332M BChE (i.e., AME-359, which has the highestcatalytic efficiency within all BChE mutants reported prior to thepresent study) against (−)-cocaine. The encouraging outcome of thisstudy suggests that the transition-state modeling is a promisingapproach for rational design of high-activity mutants of BChE as atherapeutic treatment of cocaine abuse.

Experiment 3 MD Simulation and Generation of MutantA199S/S287G/A328W/Y332G BCHE (Mutant 3) (SEQ ID NO: 14).

The following simulation and mutant generation relate toA199S/S287G/A328W/Y332G (mutant 3) as a rational design of ahigh-activity mutant of BChE against (−)-cocaine based on detailedcomputational modeling of the transition state for the rate-determiningstep (i.e., the first step of the chemical reaction process). Moleculardynamics (MD) simulations were performed to model the proteinenvironmental effects on the stabilization of the transition-statestructure for BChE-catalyzed hydrolysis of (−)-cocaine as describedabove for mutant A199S/A328W/Y332G (mutant 1). The simulated resultsindicate that the transition-state structure can be stabilized muchbetter by the protein environment in A199S/S287G/A328W/Y332G BChE thanthat in wild-type BChE and in other BChE mutants examined. Thecomputational modeling led to a prediction of the higher catalyticefficiency for the A199S/S287G/A328W/Y332G mutant against (−)-cocaine.The prediction has been confirmed by wet experimental tests showing thatthe A199S/S287G/A328W/Y332G mutant has a remarkably improved catalyticefficiency against (−)-cocaine. All of the obtained results clearlydemonstrate that directly modeling the transition-state structureprovides a reliable computational approach to the rational design of ahigh-activity mutant of BChE against (−)-cocaine.

MD simulations. As with the A199S/A328W/Y332G mutant, when performingany MD simulation on a transition state, in principle, MD simulationusing a classical force field (molecular mechanics) can only simulate astable structure corresponding to a local minimum on the potentialenergy surface, whereas a transition state during a reaction process isalways associated with a first-order saddle point on the potentialenergy surface.

The initial BChE structures used in the MD simulations were preparedbased on our previous MD simulation as above with the prior mutant andthe prereactive BChE-(−)-cocaine complex (ES) started from the X-raycrystal structure deposited in the Protein Data Bank (pdb code: 1 POP).

The general procedure for carrying out the MD simulations in water isessentially the same as that used in the computational studies formutant A199S/A328W/Y332G (mutant 1). Site-directed mutagenesis of humanBChE cDNA was performed as described before.

The MD simulation in water was performed as described above, on thismutant. Depicted in FIG. 6 are plots of four important H—O distances inthe MD-simulated TS1 structure versus the simulation time for(−)-cocaine hydrolysis catalyzed by A199S/S287G/A328W/Y332G BChE, alongwith the root-mean-square deviation (RMSD) of the simulated positions ofbackbone atoms from those in the corresponding initial structure. TracesD1, D2, and D3 refer to the distances between the carbonyl oxygen of(−)-cocaine and the NH hydrogen of G116, G117, and S199, respectively.Trace D4 is the internuclear distance between the carbonyl oxygen of(−)-cocaine and the hydroxyl hydrogen of the S199 side chain inA199S/S287G/A328W/Y332G BChE (mutant 3). RMSD represents theroot-mean-square deviation (in Å) of the simulated positions of theprotein backbone atoms from those in the initial structure.

The H—O distances in the simulated TS1 structures for wild-type BChE andits three mutants are summarized in Table 4. The H—O distances betweenthe carbonyl oxygen of (−)-cocaine and the peptidic NH hydrogen atoms ofG116, G117, and A199 (or S199) of BChE are denoted by D1, D2, and D3,respectively, in Table 4 and FIG. 6. D4 in Table 4 and FIG. 6 refers tothe H—O distance between the carbonyl oxygen of (−)-cocaine and thehydroxyl hydrogen of S199 side chain in the simulated TS1 structurecorresponding to the A199S/S287G/A328W/Y332G mutant.

TABLE 4 Summary of the MD-simulated key distances (in Å) and thecalculated total hydrogen-bonding energies (HBE, in kcal/mol) betweenthe oxyanion hole and the carbonyl oxygen of (−)-cocaine benzoyl esterin the first transition state (TS1). Distance^(a) Transition State D1 D2D3 D4 Total HBE^(b) TS1 structure for (−)-cocaine Average 4.59 2.91 1.92−5.5 (−4.6) hydrolysis catalyzed by Maximum 5.73 4.14 2.35 wild-typeBChE Minimum 3.35 1.97 1.61 Fluctuation 0.35 0.35 0.12 TS1 structure for(−)-cocaine Average 3.62 2.35 1.95 −6.2 (−4.9) hydrolysis catalyzed byMaximum 4.35 3.37 3.02 A328W/Y332A mutant of BChE Minimum 2.92 1.78 1.61Fluctuation 0.23 0.27 0.17 TS1 structure for (−)-cocaine Average 3.602.25 1.97 −6.4 (−5.0) hydrolysis catalyzed by Maximum 4.24 3.17 2.76A328W/Y332G mutant of Minimum 2.89 1.77 1.62 BChE Fluctuation 0.23 0.240.17 TS1 structure for (−)-cocaine Average 4.39 2.60 2.01 1.76 −14.0(−12.0) hydrolysis catalyzed by Maximum 5.72 4.42 2.68 2.50A199S/S287G/A328W/Y332G Minimum 2.87 1.76 1.62 1.48 mutant of BChEFluctuation 0.48 0.36 0.17 0.12 ^(a)D1, D2, and D3 represent theinternuclear distances between the carbonyl oxygen of cocaine benzoylester and the NH hydrogen of residues #116 (i.e., G116), #117 (i.e.,G117), and #199 (i.e., A199 or S199) of BChE, respectively. D4 is theinternuclear distance between the carbonyl oxygen of cocaine benzoylester and the hydroxyl hydrogen of S199 side chain in theA199S/S287G/A328W/Y332G mutant. ^(b)The total HBE value is the averageof the HBE values calculated by using the instantaneous distances in allof the snapshots. The value in parenthesis is the total HBE valuecalculated by using the MD-simulated average distances.

As seen in Table 4, the simulated H—O distance D1 is always too long forthe peptidic NH of G116 to form a N—H—O hydrogen bond with the carbonyloxygen of (−)-cocaine in all of the simulated TS1 structures. In thesimulated TS1 structure for wild-type BChE, the carbonyl oxygen of(−)-cocaine formed a firm N—H, O hydrogen bond with the peptidic NHhydrogen atom of A199 residue; the simulated H O distance (D2) was 1.61to 2.35 Å, with an average D2 value of 1.92 Å. Meanwhile, the carbonyloxygen of (−)-cocaine also had a partial N—H—O hydrogen bond with thepeptidic NH hydrogen atom of G117 residue; the simulated H—O distance(D3) was 1.97 to 4.14 Å (the average D3 value: 2.91 Å). The average D2and D3 values became 2.35 and 1.95 Å, respectively, in the simulated TS1structure for the A328W/Y332A mutant. These distances suggest a slightlyweaker N—H—O hydrogen bond with A199, but a stronger N—H—O hydrogen bondwith G117, in the simulated TS1 structure for the A328W/Y332A mutantthat the corresponding N—H—O hydrogen bonds for the wild-type. Theaverage D2 and D3 values (2.25 and 1.97 Å, respectively) in thesimulated TS1 structure for the A328W/Y332G mutant are close to thecorresponding distances for the A328W/Y332A mutant. The overall strengthof the hydrogen bonding between the carbonyl oxygen of (−)-cocaine andthe oxyanion hole of the enzyme is not expected to change considerablywhen wild-type BChE is replaced by the A328W/Y332A or A328W/Y332Gmutant.

However, the story for the simulated TS1 structure for theA199S/S287G/A328W/Y332G mutant was remarkably different. As one can seefrom Scheme 5, FIG. 6, and Table 4, when residue #199 becomes a serine(i.e., S199), the hydroxyl group on the side chain of S199 can alsohydrogen-bond to the carbonyl oxygen of (−)-cocaine to form an O—H—Ohydrogen bond, in addition to the two N—H—O hydrogen bonds with thepeptidic NH of G117 and S199. The simulated average H—O distances withthe peptidic NH hydrogen of G117, peptidic NH hydrogen of S199, andhydroxyl hydrogen of S199 are 2.60, 2.01, and 1.76 Å, respectively. Dueto the additional O—H—O hydrogen bond, the overall strength of thehydrogen bonding with the modified oxyanion hole ofA199S/S287G/A328W/Y332G BChE should be significantly stronger than thatof wild-type, A328W/Y332A, and A328W/Y332G BChE's.

Scheme 5. Schematic representation of the transition-state structure forfirst reaction step for (−)-cocaine hydrolysis catalyzed by a BChEmutant with an A199S mutation.

To better represent the overall strength of hydrogen bonding between thecarbonyl oxygen of (−)-cocaine and the oxyanion hole in a MD-simulatedTS1 structure, the hydrogen bonding energy (HBE) associated with eachsimulated H—O distance was estimated by using the empirical HBE equationimplemented in AutoDock 3.0 program suite of (a) Masson, P.; Legrand,P.; Bartels, C. F.; Froment, M.-T.; Schopfer, L. M.; Lockridge, O.Biochemistry 1997, 36, 2266, (b) Masson, P.; Xie, W., Froment, M-T.;Levitsky, V.; Fortier, P.-L.; Albaret, C.; Lockridge, O. Biochim.Biophys. Acta 1999, 1433, 281, (c) Xie, W.; Altamirano, C. V.; Bartels,C. F.; Speirs, R. J.; Cashman, J. R.; Lockridge, O. Mol. Pharmacol.1999, 55, 83, (d) Duysen, E. G.; Bartels, C. F.; Lockridge, O. J.Pharmacol. Exp. Ther. 2002, 302, 751, (e) Nachon, F.; Nicolet, Y.;Viguie, N.; Masson, P.; Fontecilla-Camps, J. C.; Lockridge, O. Eur. J.Biochem. 2002, 269, 630, herein all incorporated by reference.Specifically, for each hydrogen bond with the carbonyl oxygen of(−)-cocaine, a HBE value can be evaluated with each snapshot of theMD-simulated structure. The final HBE of the MD-simulated hydrogen bondis considered to be the average HBE value of all snapshots taken fromthe stable MD trajectory. The estimated total HBE value for the hydrogenbonds between the carbonyl oxygen of (−)-cocaine and the oxyanion holein each simulated TS1 structure is also listed in Table 4.

The HBE for each hydrogen bond was estimated by using the MD-simulatedaverage H—O distance. As seen in Table 4, the total hydrogen-bondingenergies (i.e., −4.6, −4.9, −5.0, and −12.0 kcal/mol for the wild-type,A328W/Y332A, A328W/Y332G, and A199S/S287G/A328W/Y332G BChE's,respectively) estimated in this way are systematically higher (i.e.,less negative) than the corresponding total hydrogen-bonding energies(i.e., −5.5, −6.2, −6.4, and −14.0 kcal/mol) estimated in theaforementioned way. However, the two sets of total HBE values arequalitatively consistent with each other in terms of the relativehydrogen-bonding strengths in the three simulated TS1 structures. Inparticular, the two sets of total HBE values consistently reveal thatthe overall strength of the hydrogen bonding between the carbonyl oxygenof (−)-cocaine and the oxyanion hole in the simulated TS1 structure forA199S/S287G/A328W/Y332G BChE is significantly higher than that forwild-type, A328W/Y332A, and A328W/Y332G BChE's.

Catalytic activity. The computational results discussed above providesevidence that the transition state for the first chemical reaction step(TS1) of (−)-cocaine hydrolysis catalyzed by the A199S/S287G/A328W/Y332Gmutant should be significantly more stable than that by the A328W/Y332Aor A328W/Y332G mutant, due to the significant increase of the overallhydrogen bonding between the carbonyl oxygen of (−)-cocaine and theoxyanion hole of the enzyme in the TS1 structure. The aforementionedanalysis of the literature of Sun et al and Hamza et al also indicatesthat the first chemical reaction step associated with TS1 should be therate-determining step of (−)-cocaine hydrolysis catalyzed by a BChEmutant including Y332A or Y332G mutation, although the formation of theprereactive enzyme-substrate complex (ES) is the rate-determining stepfor (−)-cocaine hydrolysis catalyzed by wild-type BChE. This suggests aclear correlation between the TS1 stabilization and the catalyticactivity of A328W/Y332A, A328W/Y332G, and A199S/S287G/A328W/Y332G BChE'sfor (−)-cocaine hydrolysis: the more stable the TS1 structure, the lowerthe energy barrier, and the higher the catalytic activity. Thus, the MDsimulations predict that A199S/S287G/A328W/Y332G BChE should have ahigher catalytic activity than A328W/Y332A or A328W/Y332G BChE for(−)-cocaine hydrolysis.

The catalytic efficiency (k_(cat)/K_(M)) of A328W/Y332A BChE for(−)-cocaine hydrolysis was reported to be ˜8.6×10⁶ M min⁻¹, which is˜9.4 times of the k_(cat)/K_(M) value (˜9.1×10⁵ M min⁻¹) of wild-typeBChE for (−)-cocaine hydrolysis. The catalytic efficiency of A328W/Y332GBChE was found to be slightly higher than that of A328W/Y332A BChE for(−)-cocaine hydrolysis. To examine the theoretical prediction of thehigher catalytic activity for A199S/S287G/A328W/Y332G BChE, theA328W/Y332A and A199S/S287G/A328W/Y332G mutants of BChE were produced aspreviously discussed through site-directed mutagenesis. To minimize thepossible systematic experimental errors of the kinetic data, kineticstudies were performed with the two mutants and wild-type BChE under thesame condition and compared the catalytic efficiency of A328W/Y332A andA199S/S287G/A328W/Y332G BChE's to that of the wild-type for (−)-cocainehydrolysis at benzoyl ester group. Based on the kinetic analysis of themeasured time-dependent radiometric data and the ELISA data, the ratioof the k_(cat)/K_(M) value of A328W/Y332A BChE to the k_(cat)/K_(M)value of wild-type BChE for (−)-cocaine hydrolysis was determined to be˜8.6. The determined catalytic efficiency ratio of ˜8.6 is in goodagreement with the ratio of ˜9.4 determined by Sun et al. Further, byusing the same experimental protocol, the ratio of the k_(cat)/K_(M)value of A199S/S287G/A328W/Y332G BChE to the k_(cat)/K_(M) value ofA328W/Y332A BChE for (−)-cocaine hydrolysis was determined to be ˜50.6.These data indicate that the ratio of the k_(cat)/K_(M) value ofA199S/S287G/A328W/Y332G BChE to the k_(cat)/K_(M) value of wild-typeBChE for (−)-cocaine hydrolysis should be ˜50.6×8.6=˜435 or˜50.6×9.4=˜476. Thus, A199S/S287G/A328W/Y332G BChE has a ˜(456±41)-foldimproved catalytic efficiency against (−)-cocaine compared to thewild-type, or A199S/S287G/A328W/Y332G BChE has a k_(cat)/K_(M) value of˜(4.15±0.37)×10⁸ M min⁻¹ for (−)-cocaine hydrolysis. The catalyticefficiency of A199S/S287G/A328W/Y332G BChE against (−)-cocaine is muchhigher than that of AME-359 (i.e., F227A/S287G/A328W/Y332M BChE,k_(cat)/K_(M)=3.1×10⁷ M min¹:, whose catalytic efficiency against(−)-cocaine is the highest within all of the previously reported BChEmutants) which has a ˜34-fold improved catalytic efficiency against(−)-cocaine compared to wild-type BChE.

By using the designed A199S/S287G/A328W/Y332G BChE as an exogenousenzyme in human, when the concentration of this mutant is kept the sameas that of the wild-type BChE in plasma, the half-life time of(−)-cocaine in plasma should be reduced from the ˜45-90 min to only˜6-12 seconds, considerably shorter than the time required for cocainecrossing the blood-brain barrier to reach CNS. Hence, the outcome ofthis study could eventually result in a valuable, efficient anti-cocainemedication.

Conclusion. The transition-state simulations demonstrate that theoverall hydrogen bonding between the carbonyl oxygen of (−)-cocainebenzoyl ester and the oxyanion hole of BChE in the TS1 structure for(−)-cocaine hydrolysis catalyzed by A199S/S287G/A328W/Y332G BChE shouldbe significantly stronger than that in the TS1 structure for (−)-cocainehydrolysis catalyzed by the wild-type BChE and other BChE mutantssimulated. Thus, the MD simulations predict that A199S/S287G/A328W/Y332GBChE should have a significantly lower energy barrier for the chemicalreaction process and, therefore, a significantly higher catalyticefficiency (k_(cat)/K_(M)) for (−)-cocaine hydrolysis. The theoreticalprediction has been confirmed by wet experimental tests showing a˜(456±41)-fold improved catalytic efficiency for A199S/S287G/A328W/Y332GBChE against (−)-cocaine compared to the wild-type BChE. Thek_(cat)/K_(M) value determined for A199S/S287G/A328W/Y332G BChE is muchhigher than the k_(cat)/K_(M) value for AME-359 (i.e.,F227A/S287G/A328W/Y332M BChE, whose catalytic efficiency against(−)-cocaine is the highest within all of the BChE mutants reportedpreviously in literature) which has a ˜34-fold improved catalyticefficiency against (−)-cocaine compared to the wild-type BChE. Theoutcome of this study provides evidence that the transition-statesimulation is a novel and unique approach for rational enzyme redesignand drug discovery.

Although the invention has been described in detail with respect topreferred embodiments thereof, it will be apparent that the invention iscapable of numerous modifications and variations, apparent to thoseskilled in the art, without departing from the spirit and scope of theinvention.

1. A butyrylcholinesterase variant peptide comprising the amino acidsequence of SEQ ID NO:
 20. 2. A pharmaceutical composition comprising: abutyrylcholinesterase variant peptide comprising the amino acid sequenceof SEQ ID NO: 20 and a suitable pharmaceutical carrier.
 3. A method oftreating a cocaine-induced condition comprising administering to anindividual an effective amount of the butyrylcholinesterase variantpolypeptide of claim 1 to lowering blood cocaine concentration.
 4. Themethod of claim 3, wherein said butyrylcholinesterase variantpolypeptide exhibits a one-hundred-fold or more increase in cocainehydrolysis catalytic efficiency compared to wild-typebutyrylcholinesterase.